Expression of HIV polypeptides and production of virus-like particles

ABSTRACT

The present invention relates to the efficient expression of HIV polypeptides in a variety of cell types, including, but not limited to, mammalian, insect, and plant cells. Synthetic expression cassettes encoding the HIV Gag-containing polypeptides are described, as are uses of the expression cassettes in applications including DNA immunization, generation of packaging cell lines, and production of Env-, tat- or Gag-containing proteins. The invention provides methods of producing Virus-Like Particles (VLPs), as well as, uses of the VLPs including, but not limited to, vehicles for the presentation of antigens and stimulation of immune response in subjects to whom the VLPs are administered.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of Ser. No. 12/026,619, filed Feb. 6,2008, which is a division of Ser. No. 10/387,336 filed Mar. 11, 2003,now issued as U.S. Pat. No. 7,348,177, which is a continuation of Ser.No. 09/475,515, filed Dec. 3, 1999, now U.S. Pat. No. 6,602,705, andwhich claims the benefit of Ser. No. 60/114,495 filed Dec. 31, 1998 andSer. No. 60/168,471 filed Dec. 1, 1999. Each of these applications isincorporated herein by reference in its entirety.

TECHNICAL FIELD

Synthetic expression cassettes encoding the HIV polypeptides (e.g.,Gag-, pol-, prot-, reverse transcriptase, Env- or tat-containingpolypeptides) are described, as are uses of the expression cassettes.The present invention relates to the efficient expression of HIVpolypeptides in a variety of cell types. Further, the invention providesmethods of producing Virus-Like Particles (VLPs), as well as, uses ofthe VLPs and high level expression of oligomeric envelope proteins.

BACKGROUND OF THE INVENTION

Acquired immune deficiency syndrome (AIDS) is recognized as one of thegreatest health threats facing modern medicine. There is, as yet, nocure for this disease.

In 1983-1984, three groups independently identified the suspectedetiological agent of AIDS. See, e.g., Barre-Sinoussi et al. (1983)Science 220:868-871; Montagnier et al., in Human T-Cell Leukemia Viruses(Gallo, Essex & Gross, eds., 1984); Vilmer et al. (1984) The Lancet1:753; Popovic et al. (1984) Science 224:497-500; Levy et al. (1984)Science 225:840-842. These isolates were variously calledlymphadenopathy-associated virus (LAV), human T-cell lymphotropic virustype III (HTLV-III), or AIDS-associated retrovirus (ARV). All of theseisolates are strains of the same virus, and were later collectivelynamed Human Immunodeficiency Virus (HIV). With the isolation of arelated AIDS-causing virus, the strains originally called HIV are nowtermed HIV-1 and the related virus is called HIV-2 See, e.g., Guyader etal. (1987) Nature 326:662-669; Brun-Vezinet et al. (1986) 233:343-346;Clavel et al. (1986) Nature 324:691-695.

A great deal of information has been gathered about the HIV virus,however, to date an effective vaccine has not been identified. Severaltargets for vaccine development have been examined including the env,Gag, poi and tat gene products encoded by HIV.

Haas, et al., (Current Biology 6(3):315-324, 1996) suggested thatselective codon usage by HIV-1 appeared to account for a substantialfraction of the inefficiency of viral protein synthesis. Andre, et al.,(J. Virol. 72(2):1497-1503, 1998) described an increased immune responseelicited by DNA vaccination employing a synthetic gp120 sequence withoptimized codon usage. Schneider, et al., (J. Virol. 71(7):4892-4903,1997) discuss inactivation of inhibitory (or instability) elements (INS)located within the coding sequences of the Gag and Gag-protease codingsequences.

The Gag proteins of HIV-1 are necessary for the assembly of virus-likeparticles. HIV-1 Gag proteins are involved in many stages of the lifecycle of the virus including, assembly, virion maturation after particlerelease, and early post-entry steps in virus replication. The roles ofHIV-1 Gag proteins are numerous and complex (Freed, E. O., Virology251:1-15, 1998).

Wolf, et al., (PCT International Application, WO 96/30523, published 3Oct. 1996; European Patent Application, Publication No. 0 449 116 A1,published 2 Oct. 1991) have described the use of altered pr55 Gag ofHIV-1 to act as a non-infectious retroviral-like particulate carrier, inparticular, for the presentation of immunologically important epitopes.Wang, et al., (Virology 200:524-534, 1994) describe a system to studyassembly of HIV Gag-β-galactosidase fusion proteins into virions. Theydescribe the construction of sequences encoding HIV Gag-β-galactosidasefusion proteins, the expression of such sequences in the presence of HIVGag proteins, and assembly of these proteins into virus particles.

Recently, Shiver, et al., (PCT International Application, WO 98/34640,published 13 Aug. 1998) described altering HIV-1 (CAM1) Gag codingsequences to produce synthetic DNA molecules encoding HIV Gag andmodifications of HIV Gag. The codons of the synthetic molecules werecodons preferred by a projected host cell.

The envelope protein of HIV-1 is a glycoprotein of about 160 kD (gp160).During virus infection of the host cell, gp160 is cleaved by host cellproteases to form gp120 and the integral membrane protein, gp41. Thegp41 portion is anchored in (and spans) the membrane bilayer of virion,while the gp120 segment protrudes into the surrounding environment. Asthere is no covalent attachment between gp120 and gp41, free gp120 isreleased from the surface of virions and infected cells.

Haas, et al., (Current Biology 6(3):315-324, 1996) suggested thatselective codon usage by HIV-1 appeared to account for a substantialfraction of the inefficiency of viral protein synthesis. Andre, et al.,(J. Virol. 72(2):1497-1503, 1998) described an increased immune responseelicited by DNA vaccination employing a synthetic gp120 sequence withoptimized codon usage.

SUMMARY OF THE INVENTION

The present invention relates to improved expression of HIV Env-, tat-,pol-, prot-, reverse transcriptase, or Gag-containing polypeptides andproduction of virus-like particles.

In one embodiment the present invention includes an expression cassette,comprising a polynucleotide encoding an HIV Gag polypeptide comprising asequence having at least 90% sequence identity to the sequence presentedas SEQ ID NO:20. In certain embodiments, the polynucleotide sequenceencoding said Gag polypeptide comprises a sequence having at least 90%sequence identity to the sequence presented as SEQ ID NO:9 or SEQ IDNO:4. The expression cassettes may further include a polynucleotidesequence encoding an HIV protease polypeptide, for example a nucleotidesequence having at least 90% sequence identity to a sequence selectedfrom the group consisting of: SEQ ID NO:5, SEQ ID NO:78, and SEQ IDNO:79. The expression cassettes may further include a polynucleotidesequence encoding an HIV reverse transcriptase polypeptide, for examplea sequence having at least 90% sequence identity to a sequence selectedfrom the group consisting of: SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82,SEQ ID NO:83, and SEQ ID NO:84. The expression cassettes may furtherinclude a polynucleotide sequence encoding an HIV tat polypeptide, forexample a sequence selected from the group consisting of: SEQ ID NO:87,SEQ ID NO:88, and SEQ ID NO:89. The expression cassettes may furtherinclude a polynucleotide sequence encoding an HIV polymerasepolypeptide, for example a sequence having at least 90% sequenceidentity to the sequence presented as SEQ ID NO:6. The expressioncassettes may include a polynucleotide sequence encoding an HIVpolymerase polypeptide, wherein (i) the nucleotide sequence encodingsaid polypeptide comprises a sequence having at least 90% sequenceidentity to the sequence presented as SEQ ID NO:4, and (ii) wherein thesequence is modified by deletions of coding regions corresponding toreverse transcriptase and integrase. The expression cassettes describedabove may preserves T-helper cell and CTL epitopes. The expressioncassettes may further include a polynucleotide sequence encoding an HCVcore polypeptide, for example a sequence having at least 90% sequenceidentity to the sequence presented as SEQ ID NO:7.

In another aspect, the invention includes an expression cassette,comprising a polynucleotide sequence encoding a polypeptide including anHIV Env polypeptide, wherein the polynucleotide sequence encoding saidEnv polypeptide comprises a sequence having at least 90% sequenceidentity to SEQ ID NO:71 (FIG. 58) or SEQ ID NO:72 (FIG. 59). In certainembodiments, the Env expression cassettes includes sequences flanking aV1 region but have a deletion in the V1 region itself, for example thesequence presented as SEQ ID NO:65 (FIG. 52, gp160.modUS4.delV1). Incertain embodiments, the Env expression cassettes, include sequencesflanking a V2 region but have a deletion in the V2 region itself, forexample the sequences shown in SEQ ID NO:60 (FIG. 47); SEQ ID NO:66(FIG. 53); SEQ ID NO:34 (FIG. 20); SEQ ID NO:37 (FIG. 24); SEQ ID NO:40(FIG. 27); SEQ ID NO:43 (FIG. 30); SEQ ID NO:46 (FIG. 33); SEQ ID NO:76(FIG. 64) and SEQ ID NO:49 (FIG. 36). In certain embodiments, the Envexpression cassettes include sequences flanking a V1/V2 region but havea deletion in the V1/V2 region itself, for example, SEQ ID NO:59 (FIG.46); SEQ ID NO:61 (FIG. 48); SEQ ID NO:67 (FIG. 54); SEQ ID NO:75 (FIG.63); SEQ ID NO:35 (FIG. 21); SEQ ID NO:38 (FIG. 25); SEQ ID NO:41 (FIG.28); SEQ ID NO:44 (FIG. 31); SEQ ID NO:47 (FIG. 34) and SEQ ID NO:50(FIG. 37). The Env-encoding expression cassettes may also include amutated cleavage site that prevents the cleavage of a gp140 polypeptideinto a gp120 polypeptide and a gp41 polypeptide, for example, SEQ IDNO:57 (FIG. 44); SEQ ID NO:61 (FIG. 48); SEQ ID NO:63 (FIG. 50); SEQ IDNO:39 (FIG. 26); SEQ ID NO:40 (FIG. 27); SEQ ID NO:41 (FIG. 28); SEQ IDNO:42 (FIG. 29); SEQ ID NO:43 (FIG. 30); SEQ ID NO:44 (FIG. 31); SEQ IDNO:45 (FIG. 32); SEQ ID NO:46 (FIG. 33); and SEQ ID NO:47 (FIG. 34). TheEnv expression cassettes may include a gp160 Env polypeptide or apolypeptide derived from a gp160 Env polypeptide, for example SEQ IDNO:64 (FIG. 51); SEQ ID NO:65 (FIG. 52); SEQ ID NO:66 (FIG. 53); SEQ IDNO:67 (FIG. 54); SEQ ID NO:68 (FIG. 55); SEQ ID NO:75 (FIG. 63); SEQ IDNO:73 (FIG. 61); SEQ ID NO:48 (FIG. 35); SEQ ID NO:49 (FIG. 36); SEQ IDNO:50 (FIG. 37); SEQ ID NO:76 (FIG. 64); and SEQ ID NO:74 (FIG. 62). TheEnv expression cassettes may include a gp140 Env polypeptide or apolypeptide derived from a gp140 Env polypeptide, for example SEQ IDNO:56 (FIG. 43); SEQ ID NO:57 (FIG. 44); SEQ ID NO:58 (FIG. 45); SEQ IDNO:59 (FIG. 46); SEQ ID NO:60 (FIG. 47); SEQ ID NO:61 (FIG. 48); SEQ IDNO:62 (FIG. 49); SEQ ID NO:63 (FIG. 50); SEQ ID NO:36 (FIG. 23); SEQ IDNO:37 (FIG. 24); SEQ ID NO:38 (FIG. 25); SEQ ID NO:39 (FIG. 26); SEQ IDNO:40 (FIG. 27); SEQ ID NO:41 (FIG. 28); SEQ ID NO:42 (FIG. 29); SEQ IDNO:43 (FIG. 30); SEQ ID NO:44 (FIG. 31); SEQ ID NO:45 (FIG. 32); SEQ IDNO:46 (FIG. 33); and SEQ ID NO:47 (FIG. 34). The Env expressioncassettes may also include a gp120 Env polypeptide or a polypeptidederived from a gp120 Env polypeptide, for example SEQ ID NO:54 (FIG.41); and SEQ ID NO:55 (FIG. 42); SEQ ID NO:33 (FIG. 19); SEQ ID NO:34(FIG. 20); and SEQ ID NO:35 (FIG. 21). The Env expression cassettes mayinclude an Env polypeptide lacking the amino acids corresponding toresidues 128 to about 194, relative to strains SF162 or US4, forexample, SEQ ID NO:55 (FIG. 42); SEQ ID NO:62 (FIG. 49); SEQ ID NO:63(FIG. 50); and SEQ ID NO:68 (FIG. 55).

In another aspect, the invention includes a recombinant expressionsystem for use in a selected host cell, comprising, one or more of theexpression cassettes described herein operably linked to controlelements compatible with expression in the selected host cell. Theexpression cassettes may be included on one or on multiple vectors andmay use the same or different promoters. Exemplary control elementsinclude a transcription promoter (e.g., CMV, CMV+intron A, SV40, RSV,HIV-Ltr, MMLV-ltr, and metallothionein), a transcription enhancerelement, a transcription termination signal, polyadenylation sequences,sequences for optimization of initiation of translation, and translationtermination sequences.

In another aspect, the invention includes a recombinant expressionsystem for use in a selected host cell, comprising, any one of theexpression cassettes described herein operably linked to controlelements compatible with expression in the selected host cell. Exemplarycontrol elements include, but are not limited to, a transcriptionpromoter (e.g., CMV, CMV+intron A, SV40, RSV, HIV-LTR, MMLV-LTR, andmetallothionein), a transcription enhancer element, a transcriptiontermination signal, polyadenylation sequences, sequences foroptimization of initiation of translation, and translation terminationsequences.

In yet another aspect, the invention includes a cell comprising one ormore of the expression cassettes described herein operably linked tocontrol elements compatible with expression in the cell. The cell canbe, for example, a mammalian cell (e.g., BHK, VERO, HT1080, 293, RD,COS-7, or CHO cells), an insect cell (e.g., Trichoplusia ni (Tn5) orSf9), a bacterial cell, a plant cell, a yeast cell, an antigenpresenting cell (e.g., primary, immortalized or tumor-derived lymphoidcells such as macrophages, monocytes, dendritic cells, B-cells, T-cells,stem cells, and progenitor cells thereof).

In another aspect, the invention includes methods for producing apolypeptide including HIV Gag-, prot-, pol-, reverse transcriptase, Env-or Tat-containing polypeptide sequences, said method comprising,incubating the cells comprising one or more the expression cassettesdescribe herein, under conditions for producing said polypeptide.

In yet another aspect, the invention includes compositions forgenerating an immunological response, comprising one or more of theexpression cassettes described herein. In certain embodiments, thecompositions also include an adjuvant.

In a still further aspect, the invention includes methods of generatingan immune response in a subject, comprising introducing a compositioncomprising one or more of the expression cassettes described herein intothe subject under conditions that are compatible with expression of saidexpression cassette in the subject. In certain embodiments, theexpression cassette is introduced using a gene delivery vector. Morethan one expression cassette may be introduced using one or more genedelivery vectors.

In yet another aspect, the invention includes a purified polynucleotidecomprising a polynucleotide sequence encoding a polypeptide including anHIV Env polypeptide, wherein the polynucleotide sequence encoding saidEnv polypeptide comprises a sequence having at least 90% sequenceidentity to SEQ ID NO:71 (FIG. 58) or SEQ ID NO:72 (FIG. 59). Furtherexemplary purified polynucleotide sequences were presented above.

The polynucleotides of the present invention can be produced byrecombinant techniques, synthetic techniques, or combinations thereof.

In another embodiment, the invention includes a method for producing apolypeptide including HIV Gag polypeptide sequences, where the methodcomprises incubating any of the above cells containing an expressioncassette of interest under conditions for producing the polypeptide.

The invention further includes, a method for producing virus-likeparticles (VLPs) where the method comprises incubating any of theabove-described cells containing an expression cassette of interestunder conditions for producing VLPs.

In another aspect the invention includes a method for producing acomposition of virus-like particles (VLPs) where, any of theabove-described cells containing an expression cassette of interest areincubated under conditions for producing VLPs, and the VLPs aresubstantially purified to produce a composition of VLPs.

In a further embodiment of the present invention, packaging cell linesare produced using the expression cassettes of the present invention.For example, a cell line useful for packaging lentivirus vectorscomprises suitable host cells that have an expression vector containingan expression cassette of the present invention wherein saidpolynucleotide sequence is operably linked to control elementscompatible with expression in the host cell. In a preferred embodiment,such host cells may be transfected with one or more expression cassetteshaving a polynucleotide sequence that encodes an HIV polymerasepolypeptide or polypeptides derived therefrom, for example, where thenucleotide sequence encoding said polypeptide comprises a sequencehaving at least 90% sequence identity to the sequence presented as SEQID NO:6. Further, the HIV polymerase polypeptide may be modified bydeletions of coding regions corresponding to reverse transcriptase andintegrase. Such a polynucleotide sequence may preserve T-helper cell andCTL epitopes, for example when used in a vaccine application. Inaddition, the polynucleotide sequence may also include otherpolypeptides. Further, polynucleotide sequences encoding additionalpolypeptides whose expression are useful for packaging cell linefunction may also be utilized.

In another aspect, the present invention includes a gene delivery orvaccine vector for use in a subject, where the vector is a suitable genedelivery vector for use in the subject, and the vector comprises one ormore of any of the expression cassettes of the present invention wherethe polynucleotide sequences of interest are operably linked to controlelements compatible with expression in the subject. Such gene deliveryvectors can be used in a method of DNA immunization of a subject, forexample, by introducing a gene delivery vector into the subject underconditions that are compatible with expression of the expressioncassette in the subject. Gene delivery vectors useful in the practice ofthe present invention include, but are not limited to, nonviral vectors,bacterial plasmid vectors, viral vectors, particulate carriers (wherethe vector is coated on a polylactide co-glycolide particles, gold ortungsten particle, for example, the coated particle can be delivered toa subject cell using a gene gun), liposome preparations, and viralvectors (e.g., vectors derived from alphaviruses, pox viruses, andvaccinia viruses, as well as, retroviral vectors, including, but notlimited to, lentiviral vectors). Alphavirus-derived vectors include, forexample, an alphavirus cDNA construct, a recombinant alphavirus particlepreparation and a eukaryotic layered vector initiation system. In oneembodiment, the subject is a vertebrate, preferably a mammal, and in afurther embodiment the subject is a human.

The invention further includes a method of generating an immune responsein a subject, where cells of a subject are transfected with any of theabove-described gene delivery vectors (e.g., alphavirus constructs;alphavirus cDNA constructs; eukaryotic layered vector initiation systems(see, e.g., U.S. Pat. No. 5,814,482 for description of suitableeukaryotic layered vector initiation systems); alphavirus particlepreparations; etc.) under conditions that permit the expression of aselected polynucleotide and production of a polypeptide of interest(i.e., encoded by any expression cassette of the present invention),thereby eliciting an immunological response to the polypeptide.Transfection of the cells may be performed ex vivo and the transfectedcells are reintroduced into the subject. Alternately, or in addition,the cells may be transfected in vivo in the subject. The immune responsemay be humoral and/or cell-mediated (cellular).

Further embodiments of the present invention include purifiedpolynucleotides. In one embodiment, the purified polynucleotidecomprises a polynucleotide sequence having at least 90% sequenceidentity to the sequence presented as SEQ ID NO:20, and complementsthereof. In another embodiment, the purified polynucleotide comprises apolynucleotide sequence encoding an HIV Gag polypeptide, wherein thepolynucleotide sequence comprises a sequence having at least 90%sequence identity to the sequence presented as SEQ ID NO:20, andcomplements thereof. In still another embodiment, the purifiedpolynucleotide comprises a polynucleotide sequence encoding an HIV Gagpolypeptide, wherein the polynucleotide sequence comprises a sequencehaving at least 90% sequence identity to the sequence presented as SEQID NO:9, and complements thereof. In further embodiments thepolynucleotide sequence comprises a sequence having at least 90%sequence identity to one of the following sequences: SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, and complements thereof.

The polynucleotides of the present invention can be produced byrecombinant techniques, synthetic techniques, or combinations thereof.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the locations of the inactivation sites for the nativeHIV-1SF2 Gag protein coding sequence SEQ ID NO:1.

FIG. 2 shows the locations of the inactivation sites for the nativeHIV-1SF2 Gag-protease protein coding sequence SEQ ID NO:2.

FIGS. 3A and 3B show electron micrographs of virus-like particles. FIG.3A shows immature p55Gag virus-like particles in COS-7 cells transfectedwith a synthetic HIV-1_(SF2) gag construct while FIG. 3B shows mature(arrows) and immature VLP in cells transfected with a modifiedHIV-1_(SF2) gagprotease construct (GP2, SEQ ID NO:70). Transfected cellswere fixed at 24 h (gag) or 48 h (gagprotease) post-transfection andsubsequently analyzed by electron microscopy (magnification at100,000×). Cells transfected with vector alone (pCMVKm2) served asnegative control (data not shown).

FIG. 4 presents an image of samples from a series of fractions whichwere electrophoresed on an 8-16% SDS polyacrylamide gel and theresulting bands visualized by commassie blue staining. The results showthat the native p55 Gag virus-like particles (VLPs) banded at a sucrosedensity of range of 1.15-1.19 g/ml with the peak at approximately 1.17g/ml.

FIG. 5 presents an image similar to FIG. 4 where the analysis wasperformed using Gag VLPs produced by a synthetic Gag expressioncassette.

FIG. 6 presents a comparison of the total amount of purified HIV p55 Gagfrom several preparations obtained from two baculovirus expressioncassettes encoding native and modified Gag.

FIG. 7 presents an alignment of modified coding sequences of the presentinvention including a synthetic Gag expression cassette (SEQ ID NO:4), asynthetic Gag-protease expression cassette (SEQ ID NO:5), and asynthetic Gag-polymerase expression cassette (SEQ ID NO:6). A commonregion (Gag-common; SEQ ID NO:9) extends from position 1 to position1262.

FIG. 8 presents an image of wild-type Gag-HCV core expression samplesfrom a series of fractions which were electrophoresed on an 8-16% SDSpolyacrylamide gel and the resulting bands visualized by commassiestaining.

FIG. 9 shows the results of Western blot analysis of the gel shownpresented in FIG. 8.

FIG. 10 presents results similar to those shown in FIG. 9. The resultsin FIG. 10 indicate that the main HCV Core-specific reactivity migratesat an approximate molecular weight of 72,000 kD, which is in accordancewith the predicted molecular weight of the Gag-HCV core chimericprotein.

FIGS. 11A to 11D present a comparison of AT content, in percent, ofcDNAs corresponding to an unstable human mRNA (human IFNγ mRNA; 11A),wild-type HIV Gag native RNA (11B), a stable human mRNA (human GAPDHmRNA; 11C), and synthetic HIV Gag RNA (11D).

FIG. 12 shows the location of the inactivation sites for the nativeHIV-1SF2 Gag-polymerase sequence SEQ ID NO:3.

FIG. 13A presents a vector map of pESN2dhfr.

FIG. 13B presents a map of the pCMVIII vector.

FIG. 14 presents a vector map of pCMV-LINK.

FIG. 15 presents a schematic diagram showing the relationships betweenthe following forms of the HIV Env polypeptide: gp160, gp140, gp120, andgp41.

FIG. 16 depicts the nucleotide sequence of wild-type gp120 from SF162(SEQ ID NO:30).

FIG. 17 depicts the nucleotide sequence of the wild-type gp140 fromSF162 (SEQ ID NO:31).

FIG. 18 depicts the nucleotide sequence of the wild-type gp160 fromSF162 (SEQ ID NO:32).

FIG. 19 depicts the nucleotide sequence of the construct designatedgp120.modSF162 (SEQ ID NO:33).

FIG. 20 depicts the nucleotide sequence of the construct designatedgp120.modSF162.delV2 (SEQ ID NO:34).

FIG. 21 depicts the nucleotide sequence of the construct designatedgp120.modSF162.delV1/V2 (SEQ ID NO:35).

FIGS. 22A-H show the percent A-T content over the length of thesequences for IFNγ (FIGS. 2C and 2G); native gp160 Env US4 and SF162(FIGS. 2A and 2E, respectively); GAPDH (FIGS. 2D and 2H); and thesynthetic gp160 Env for US4 and SF162 (FIGS. 2B and 2F, respectively).

FIG. 23 depicts the nucleotide sequence of the construct designatedgp140.modSF162 (SEQ ID NO:36).

FIG. 24 depicts the nucleotide sequence of the construct designatedgp140.modSF162.delV2 (SEQ ID NO:37).

FIG. 25 depicts the nucleotide sequence of the construct designatedgp140.modSF162.delV1/V2 (SEQ ID NO:38).

FIG. 26 depicts the nucleotide sequence of the construct designatedgp140.mut.modSF162 (SEQ ID NO:39).

FIG. 27 depicts the nucleotide sequence of the construct designatedgp140.mut.modSF162.delV2 (SEQ ID NO:40).

FIG. 28 depicts the nucleotide sequence of the construct designatedgp140.mut.modSF162.delV1/V2 (SEQ ID NO:41).

FIG. 29 depicts the nucleotide sequence of the construct designatedgp140.mut7.modSF162 (SEQ ID NO:42).

FIG. 30 depicts the nucleotide sequence of the construct designatedgp140.mut7.modSF162.delV2 (SEQ ID NO:43).

FIG. 31 depicts the nucleotide sequence of the construct designatedgp140.mut7.modSF162.delV1/V2 (SEQ ID NO:44).

FIG. 32 depicts the nucleotide sequence of the construct designatedgp140.mut8.modSF162 (SEQ ID NO:45).

FIG. 33 depicts the nucleotide sequence of the construct designatedgp140.mut8.modSF162.delV2 (SEQ ID NO:46).

FIG. 34 depicts the nucleotide sequence of the construct designatedgp140.mut8.modSF162.delV1/V2 (SEQ ID NO:47).

FIG. 35 depicts the nucleotide sequence of the construct designatedgp160.modSF162 (SEQ ID NO:48).

FIG. 36 depicts the nucleotide sequence of the construct designatedgp160.modSF162.delV2 (SEQ ID NO:49).

FIG. 37 depicts the nucleotide sequence of the construct designatedgp160.modSF162.delV1/V2 (SEQ ID NO:50).

FIG. 38 depicts the nucleotide sequence of the wild-type gp120 from US4(SEQ ID NO:51).

FIG. 39 depicts the nucleotide sequence of the wild-type gp140 from US4(SEQ ID NO:52).

FIG. 40 depicts the nucleotide sequence of the wild-type gp160 from US4(SEQ ID NO:53).

FIG. 41 depicts the nucleotide sequence of the construct designatedgp120.modUS4 (SEQ ID NO:54).

FIG. 42 depicts the nucleotide sequence of the construct designatedgp120.modUS4.del 128-194 (SEQ ID NO:55).

FIG. 43 depicts the nucleotide sequence of the construct designatedgp140.modUS4 (SEQ ID NO:56).

FIG. 44 depicts the nucleotide sequence of the construct designatedgp140.mut.modUS4 (SEQ ID NO:57).

FIG. 45 depicts the nucleotide sequence of the construct designatedgp140.TM.modUS4 (SEQ ID NO:58).

FIG. 46 depicts the nucleotide sequence of the construct designatedgp140.modUS4.delV1/V2 (SEQ ID NO:59).

FIG. 47 depicts the nucleotide sequence of the construct designatedgp140.modUS4.delV2 (SEQ ID NO:60).

FIG. 48 depicts the nucleotide sequence of the construct designatedgp140.mut.modUS4.delV1/V2 (SEQ ID NO:61).

FIG. 49 depicts the nucleotide sequence of the construct designatedgp140.modUS4.del 128-194 (SEQ ID NO:62).

FIG. 50 depicts the nucleotide sequence of the construct designatedgp140.mut.modUS4.del 128-194 (SEQ ID NO:63).

FIG. 51 depicts the nucleotide sequence of the construct designatedgp160.modUS4 (SEQ ID NO:64).

FIG. 52 depicts the nucleotide sequence of the construct designatedgp160.modUS4.delV1 (SEQ ID NO:65).

FIG. 53 depicts the nucleotide sequence of the construct designatedgp160.modUS4.delV2 (SEQ ID NO:66).

FIG. 54 depicts the nucleotide sequence of the construct designatedgp160.modUS4.delV1/V2 (SEQ ID NO:67).

FIG. 55 depicts the nucleotide sequence of the construct designatedgp160.modUS4.del 128-194 (SEQ ID NO:68).

FIG. 56 depicts the nucleotide sequence of the common region of Env fromwild-type US4 (SEQ ID NO:69).

FIG. 57 depicts the nucleotide sequence of the common region of Env fromwild-type SF162 (SEQ ID NO:70).

FIG. 58 depicts the nucleotide sequence of synthetic sequencescorresponding to the common region of Env from US4 (SEQ ID NO:71).

FIG. 59 depicts the nucleotide sequence of synthetic sequencescorresponding to the common region of Env from SF162 (SEQ ID NO:72).

FIG. 60 presents a schematic representation of an Env polypeptidepurification strategy.

FIG. 61 depicts the nucleotide sequence of the bicistronic constructdesignated gp160.modUS4.Gag.modSF2 (SEQ ID NO:73).

FIG. 62 depicts the nucleotide sequence of the bicistronic constructdesignated gp160.modSF162.Gag.modSF2 (SEQ ID NO:74).

FIG. 63 depicts the nucleotide sequence of the bicistronic constructdesignated gp160.modUS4.-delV1/V2.Gag.modSF2 (SEQ ID NO:75).

FIG. 64 depicts the nucleotide sequence of the bicistronic constructdesignated gp160.modSF162.delV2.Gag.modSF2 (SEQ ID NO:76).

FIGS. 65A-65F show micrographs of 293T cells transfected with thefollowing polypeptide encoding sequences: FIG. 65A, gag.modSF2; FIG.65B, gp160.modUS4; FIG. 65C, gp160.modUS4.delV1/V2.gag.modSF2(bicistronic Env and Gag); FIGS. 65D and 65E, gp160.modUS4.delV1/V2 andgag.modSF2; and FIG. 65F, gp120.modSF162.delV2 and gag.modSF2.

FIGS. 66A and 66B present alignments of selected modified codingsequences of the present invention including a common region defined foreach group of synthetic Env expression cassettes. FIG. 66A presentsalignments of modified SF162 sequences. gp160.modSF162, SEQ ID NO:48;gp160.modSF162.delV2, SEQ ID NO:49; gp160.modSF162.delV1V2, SEQ IDNO:50; gp140.mut.modSF162, SEQ ID NO:39; gp140.mut7.modSF162, SEQ IDNO:42; gp140.mut8.modSF162, SEQ ID NO:45; gp120.modSF162, SEQ ID NO:33.FIG. 66B presents alignments of modified US4 sequences. gp160, SEQ IDNO:53; gp160 del V1, SEQ ID NO:65; gp160 del V2, SEQ ID NO:66; gp160 del128-194, SEQ ID NO:63; gp140™, SEQ ID NO:58; gp140, SEQ ID NO:52;gp140mut, SEQ ID NO:57; gp120, SEQ ID NO:51. The SEQ ID NOs for thesesequences are presented in Tables 1A and 1B.

FIG. 67 shows the ELISA titers (binding antibodies) obtained in tworhesus macaques (H445, lines with solid black dots; and J408, lines withopen squares). The y-axis is the end-point gp140 ELISA titers and thex-axis shows weeks post-immunization. The dashed lines at 0, 4, and 8weeks represent DNA immunizations. The alternating dash/dotted line at27 weeks indicates a DNA plus protein boost immunization.

FIG. 68 (SEQ ID NO:77) depicts the wild-type nucleotide sequence of Gagreverse transcriptase from SF2.

FIG. 69 (SEQ ID NO:78) depicts the nucleotide sequence of the constructdesignated GP1.

FIG. 70 (SEQ ID NO:79) depicts the nucleotide sequence of the constructdesignated GP2.

FIG. 71 (SEQ ID NO:80) depicts the nucleotide sequence of the constructdesignated FS(+).protinact_RTopt.YM. FS(+) indicates that there is aframeshift in the GagPol coding sequence.

FIG. 72 (SEQ ID NO:81) depicts the nucleotide sequence of the constructdesignated FS(+).protinact.RTopt.YMWM.

FIG. 73 (SEQ ID NO:82) depicts the nucleotide sequence of the constructdesignated FS(−).protmod.RTopt.YM. FS(−) indicates that there is noframeshift in the GagPol coding sequence.

FIG. 74 (SEQ ID NO:83) depicts the nucleotide sequence of the constructdesignated FS(−).protmod.RTopt.YMWM.

FIG. 75 (SEQ ID NO:84) depicts the nucleotide sequence of the constructdesignated FS(−).protmod.RTopt(+).

FIG. 76 (SEQ ID NO:85) depicts the nucleotide sequence of wild type Tatfrom isolate SF162.

FIG. 77 (SEQ ID NO:86) depicts the amino acid sequence of the tatpolypeptide.

FIG. 78 (SEQ ID NO:87) depicts the nucleotide sequence of a syntheticTat construct designated Tat.SF162.opt.

FIG. 79 (SEQ ID NO:88) depicts the nucleotide sequence of a syntheticTat construct designated tat.cys22.sf162.opt. The construct encodes atat polypeptide in which the cystein residue at position 22 of the wildtype Tat polypeptide is replaced by a glycine residue.

FIGS. 80A to 80E are an alignment of the nucleotide sequences of theconstructs designated Gag.mod.SF2, GP1 (SEQ ID NO:78), and GP2 (SEQ IDNO:79).

FIG. 81 (SEQ ID NO:89) depicts the nucleotide sequence of the constructdesignated tataminoSF162.opt, which encodes the amino terminus of thattat protein. The codon encoding the cystein-22 residue is underlined.

FIG. 82 (SEQ ID NO:90) depicts the amino acid sequence of thepolypeptide encoded by the construct designated tat.cys22.5F162.opt (SEQID NO:88).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed.(Ausubel et al. eds., 1999, John Wiley & Sons); Molecular BiologyTechniques: An Intensive Laboratory Course, (Ream et al., eds., 1998,Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed.(Newton & Graham eds., 1997, Springer Verlag).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “an antigen”includes a mixture of two or more such agents.

1. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Synthetic” sequences, as used herein, refers to Env-, tat- orGag-encoding polynucleotides whose expression has been optimized asdescribed herein, for example, by codon substitution, deletions,replacements and/or inactivation of inhibitory sequences. “Wild-type” or“native” sequences, as used herein, refers to polypeptide encodingsequences that are essentially as they are found in nature, e.g., Gagencoding sequences as found in the isolate HIV-1SF2 or Env encodingsequences as found in the isolates HIV-1SF162 or HIV1US4.

As used herein, the term “virus-like particle” or “VLP” refers to anonreplicating, viral shell, derived from any of several virusesdiscussed further below. VLPs are generally composed of one or moreviral proteins, such as, but not limited to those proteins referred toas capsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs canforth spontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art and discussed more fully below. The presence of VLPsfollowing recombinant expression of viral proteins can be detected usingconventional techniques known in the art, such as by electronmicroscopy, biophysical characterization, and the like. See, e.g., Bakeret al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol.(1994) 68:4503-4505. For example, VLPs can be isolated by densitygradient centrifugation and/or identified by characteristic densitybanding (e.g., Example 7). Alternatively, cryoelectron microscopy can beperformed on vitrified aqueous samples of the VLP preparation inquestion, and images recorded under appropriate exposure conditions.

By “particle-forming polypeptide” derived from a particular viralprotein is meant a full-length or near full-length viral protein, aswell as a fragment thereof, or a viral protein with internal deletions,which has the ability to form VLPs under conditions that favor VLPformation. Accordingly, the polypeptide may comprise the full-lengthsequence, fragments, truncated and partial sequences, as well as analogsand precursor forms of the reference molecule. The term thereforeintends deletions, additions and substitutions to the sequence, so longas the polypeptide retains the ability to form a VLP. Thus, the termincludes natural variations of the specified polypeptide sincevariations in coat proteins often occur between viral isolates. The termalso includes deletions, additions and substitutions that do notnaturally occur in the reference protein, so long as the protein retainsthe ability to form a VLP. Preferred substitutions are those which areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cystine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the term “immunogen.”Normally, a B-cell epitope will include at least about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term “antigen” denotes both subunit antigens, (i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature), as well as, killed,attenuated or inactivated bacteria, viruses, fungi, parasites or othermicrobes. Antibodies such as anti-idiotype antibodies, or fragmentsthereof, and synthetic peptide mimotopes, which can mimic an antigen orantigenic determinant, are also captured under the definition of antigenas used herein. Similarly, an oligonucleotide or polynucleotide whichexpresses an antigen or antigenic determinant in vivo, such as in genetherapy and DNA immunization applications, is also included in thedefinition of antigen herein.

For purposes of the present invention, antigens can be derived from anyof several known viruses, bacteria, parasites and fungi, as describedmore fully below. The term also intends any of the various tumorantigens. Furthermore, for purposes of the present invention, an“antigen” refers to a protein which includes modifications, such asdeletions, additions and substitutions (generally conservative innature), to the native sequence, so long as the protein maintains theability to elicit an immunological response, as defined herein. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations, or by measurementof epitope specific T-cells (e.g., by the tetramer technique)(reviewedby McMichael, A. J., and O'Callaghan, C. A., J. Exp. Med.187(9)1367-1371, 1998; Mcheyzer-Williams, M. G., et al, Immunol. Rev.150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865, 1997).

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest.

By “subunit vaccine” is meant a vaccine composition which includes oneor more selected antigens but not all antigens, derived from orhomologous to, an antigen from a pathogen of interest such as from avirus, bacterium, parasite or fungus. Such a composition issubstantially free of intact pathogen cells or pathogenic particles, orthe lysate of such cells or particles. Thus, a “subunit vaccine” can beprepared from at least partially purified (preferably substantiallypurified) immunogenic polypeptides from the pathogen, or analogsthereof. The method of obtaining an antigen included in the subunitvaccine can thus include standard purification techniques, recombinantproduction, or synthetic production.

“Substantially purified” general refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA,genomic DNA sequences from viral or procaryotic DNA, and even syntheticDNA sequences. A transcription termination sequence may be located 3′ tothe coding sequence.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences, see e.g., McCaughan et al. (1995) PNAS USA92:5431-5435; Kochetov et al (1998) FEBS Letts. 440:351-355.

A “nucleic acid” molecule can include, but is not limited to,procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA,genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “re-combinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting procaryotic microorganisms or eucaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An implementation of this algorithm for nucleic acid and peptidesequences is provided by the Genetics Computer Group (Madison, Wis.) intheir BestFit utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). Other equally suitable programs for calculating thepercent identity or similarity between sequences are generally known inthe art.

For example, percent identity of a particular nucleotide sequence to areference sequence can be determined using the homology algorithm ofSmith and Waterman with a default scoring table and a gap penalty of sixnucleotide positions. Another method of establishing percent identity inthe context of the present invention is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages, theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated,the “Match” value reflects “sequence identity.” Other suitable programsfor calculating the percent identity or similarity between sequences aregenerally known in the art, such as the alignment program BLAST, whichcan also be used with default parameters. For example, BLASTN and BLASTPcan be used with the following default parameters: geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address:http://www.ncbi.nlm.gov/cgi-bin/BLAST.

One of skill in the art can readily determine the proper searchparameters to use for a given sequence in the above programs. Forexample, the search parameters may vary based on the size of thesequence in question. Thus, for example, a representative embodiment ofthe present invention would include an isolated polynucleotide having Xcontiguous nucleotides, wherein (i) the X contiguous nucleotides have atleast about 50% identity to Y contiguous nucleotides derived from any ofthe sequences described herein, (ii) X equals Y, and (iii) X is greaterthan or equal to 6 nucleotides and up to 5000 nucleotides, preferablygreater than or equal to 8 nucleotides and up to 5000 nucleotides, morepreferably 10-12 nucleotides and up to 5000 nucleotides, and even morepreferably 15-20 nucleotides, up to the number of nucleotides present inthe full-length sequences described herein (e.g., see the SequenceListing and claims), including all integer values falling within theabove-described ranges.

The synthetic expression cassettes (and purified polynucleotides) of thepresent invention include related polynucleotide sequences having about80% to 100%, greater than 80-85%, preferably greater than 90-92%, morepreferably greater than 95%, and most preferably greater than 98%sequence (including all integer values falling within these describedranges) identity to the synthetic expression cassette sequencesdisclosed herein (for example, to the sequences presented in Tables 1Aand 1B) when the sequences of the present invention are used as thequery sequence.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N. Y.). Such assays can be conducted using varyingdegrees of selectivity, for example, using conditions varying from lowto high stringency. If conditions of low stringency are employed, theabsence of non-specific binding can be assessed using a secondary probethat lacks even a partial degree of sequence identity (for example, aprobe having less than about 30% sequence identity with the targetmolecule), such that, in the absence of non-specific binding events, thesecondary probe will not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” typicallyhybridizes under conditions that allow detection of a target nucleicacid sequence of at least about 10-14 nucleotides in length having atleast approximately 70% sequence identity with the sequence of theselected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N. Y.).

A first polynucleotide is “derived from” second polynucleotide if it hasthe same or substantially the same basepair sequence as a region of thesecond polynucleotide, its cDNA, complements thereof, or if it displayssequence identity as described above.

A first polypeptide is “derived from” a second polypeptide if it is (i)encoded by a first polynucleotide derived from a second polynucleotide,or (ii) displays sequence identity to the second polypeptides asdescribed above.

Generally, a viral polypeptide is “derived from” a particularpolypeptide of a virus (viral polypeptide) if it is (i) encoded by anopen reading frame of a polynucleotide of that virus (viralpolynucleotide), or (ii) displays sequence identity to polypeptides ofthat virus as described above.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences which areimmunologically identifiable with a polypeptide encoded by the sequence.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected antigens into a host cell,for the in vivo expression of an antigen, antigens, an epitope, orepitopes. The nucleic acid molecule can be introduced directly into arecipient subject, such as by injection, inhalation, oral, intranasaland mucosal administration, or the like, or can be introduced ex vivo,into cells which have been removed from the host. In the latter case,the transformed cells are reintroduced into the subject where an immuneresponse can be mounted against the antigen encoded by the nucleic acidmolecule.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting DNA or RNA of interest into a host cell. Such methodscan result in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene delivery expression vectors include,but are not limited to, vectors derived from bacterial plasmid vectors,viral vectors, non-viral vectors, alphaviruses, pox viruses and vacciniaviruses. When used for immunization, such gene delivery expressionvectors may be referred to as vaccines or vaccine vectors.

“T lymphocytes” or “T cells” are non-antibody producing lymphocytes thatconstitute a part of the cell-mediated arm of the immune system. T cellsarise from immature lymphocytes that migrate from the bone marrow to thethymus, where they undergo a maturation process under the direction ofthymic hormones. Here, the mature lymphocytes rapidly divide increasingto very large numbers. The maturing T cells become immunocompetent basedon their ability to recognize and bind a specific antigen. Activation ofimmunocompetent T cells is triggered when an antigen binds to thelymphocyte's surface receptors.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells. The term refers to both stableand transient uptake of the genetic material, and includes uptake ofpeptide- or antibody-linked DNAs.

A “vector” is capable of transferring gene sequences to target cells(e.g., bacterial plasmid vectors, viral vectors, non-viral vectors,particulate carriers, and liposomes). Typically, “vector construct,”“expression vector,” and “gene transfer vector,” mean any nucleic acidconstruct capable of directing the expression of a gene of interest andwhich can transfer gene sequences to target cells. Thus, the termincludes cloning and expression vehicles, as well as viral vectors.

Transfer of a “suicide gene” (e.g., a drug-susceptibility gene) to atarget cell renders the cell sensitive to compounds or compositions thatare relatively nontoxic to normal cells. Moolten, F. L. (1994) CancerGene Ther. 1:279-287. Examples of suicide genes are thymidine kinase ofherpes simplex virus (HSV-tk), cytochrome P450 (Manome et al. (1996)Gene Therapy 3:513-520), human deoxycytidine kinase (Manome et al.(1996) Nature Medicine 2(5):567-573) and the bacterial enzyme cytosinedeaminase (Dong et al. (1996) Human Gene Therapy 7:713-720). Cells whichexpress these genes are rendered sensitive to the effects of therelatively nontoxic prodrugs ganciclovir (HSV-tk), cyclophosphamide(cytochrome P450 2B1), cytosine arabinoside (human deoxycytidine kinase)or 5-fluorocytosine (bacterial cytosine deaminase). Culver et al. (1992)Science 256:1550-1552, Huber et al. (1994) Proc. Natl. Acad. Sci. USA91:8302-8306.

A “selectable marker” or “reporter marker” refers to a nucleotidesequence included in a gene transfer vector that has no therapeuticactivity, but rather is included to allow for simpler preparation,manufacturing, characterization or testing of the gene transfer vector.

A “specific binding agent” refers to a member of a specific binding pairof molecules wherein one of the molecules specifically binds to thesecond molecule through chemical and/or physical means. One example of aspecific binding agent is an antibody directed against a selectedantigen.

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,both adult and newborn individuals are intended to be covered. Thesystem described above is intended for use in any of the abovevertebrate species, since the immune systems of all of these vertebratesoperate similarly.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, “treatment” refers to any of (I) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

“Lentiviral vector”, and “recombinant lentiviral vector” are derivedfrom the subset of retroviral vectors known as lentiviruses. Lentiviralvectors refer to a nucleic acid construct which carries, and withincertain embodiments, is capable of directing the expression of a nucleicacid molecule of interest. The lentiviral vector includes at least onetranscriptional promoter/enhancer or locus defining element(s), or otherelements which control gene expression by other means such as alternatesplicing, nuclear RNA export, post-translational modification ofmessenger, or post-transcriptional modification of protein. Such vectorconstructs must also include a packaging signal, long terminal repeats(LTRS) or portion thereof, and positive and negative strand primerbinding sites appropriate to the lentiviral vector used (if these arenot already present in the retroviral vector). Optionally, therecombinant lentiviral vector may also include a signal which directspolyadenylation, selectable markers such as Neo, TK, hygromycin,phleomycin, histidinol, or DHFR, as well as one or more restrictionsites and a translation termination sequence. By way of example, suchvectors typically include a 5′ LTR, a tRNA binding site, a packagingsignal, an origin of second strand DNA synthesis, and a 3′LTR or aportion thereof.

“Lentiviral vector particle” as utilized within the present inventionrefers to a lentivirus which carries at least one gene of interest. Theretrovirus may also contain a selectable marker. The recombinantlentivirus is capable of reverse transcribing its genetic material (RNA)into DNA and incorporating this genetic material into a host cell's DNAupon infection. Lentiviral vector particles may have a lentiviralenvelope, a non-lentiviral envelope (e.g., an ampho or VSV-G envelope),or a chimeric envelope.

“Nucleic acid expression vector” or “Expression cassette” refers to anassembly which is capable of directing the expression of a sequence orgene of interest. The nucleic acid expression vector includes a promoterwhich is operably linked to the sequences or gene(s) of interest. Othercontrol elements may be present as well. Expression cassettes describedherein may be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude a bacterial origin of replication, one or more selectablemarkers, a signal which allows the plasmid construct to exist assingle-stranded DNA (e.g., a M13 origin of replication), a multiplecloning site, and a “mammalian” origin of replication (e.g., a SV40 oradenovirus origin of replication).

“Packaging cell” refers to a cell which contains those elementsnecessary for production of infectious recombinant retrovirus (e.g.,lentivirus) which are lacking in a recombinant retroviral vector.Typically, such packaging cells contain one or more expression cassetteswhich are capable of expressing proteins which encode Gag, pol and envproteins.

“Producer cell” or “vector producing cell” refers to a cell whichcontains all elements necessary for production of recombinant retroviralvector particles.

2. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

2.1 Synthetic Expression Cassettes

2.1.1 Modification of HIV-1 Gag Nucleic Acid Coding Sequences

One aspect of the present invention is the generation of HIV-1 Gagprotein coding sequences, and related sequences, having improvedexpression relative to the corresponding wild-type sequence. Anexemplary embodiment of the present invention is illustrated hereinmodifying the Gag protein wild-type sequences obtained from the HIV-1SF2strain (SEQ ID NO:1; Sanchez-Pescador, R., et al., Science 227(4686):484-492, 1985; Luciw, P. A., et al. U.S. Pat. No. 5,156,949, issued Oct.20, 1992, herein incorporated by reference; Luciw, P. A., et al., U.S.Pat. No. 5,688,688, Nov. 18, 1997, herein incorporated by reference).Gag sequence obtained from other HIV variants may be manipulated insimilar fashion following the teachings of the present specification.Such other variants include, but are not limited to, Gag proteinencoding sequences obtained from the isolates. HIV_(IIIb), HIV_(SF2),HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV), HIV_(LAI), HIV_(MN),HIV-1_(CM235), HIV-1_(US4), other HIV-1 strains from diverse subtypes(e.g., subtypes, A through G, and O), HIV-2 strains and diverse subtypes(e.g., HIV-2_(UC1) and HIV-2_(UC2)), and simian immunodeficiency virus(Sly). (See, e.g., Virology, 3rd Edition (W. K. Joklik ed. 1988);Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds.1991); Virology, 3rd Edition (Fields, B N, D M Knipe, P M Howley,Editors, 1996, Lippincott-Raven, Philadelphia, Pa.; for a description ofthese and other related viruses).

First, the HIV-1 codon usage pattern was modified so that the resultingnucleic acid coding sequence was comparable to codon usage found inhighly expressed human genes (Example 1). The HIV codon usage reflects ahigh content of the nucleotides A or T of the codon-triplet. The effectof the HIV-1 codon usage is a high AT content in the DNA sequence thatresults in a decreased translation ability and instability of the mRNA.In comparison, highly expressed human codons prefer the nucleotides G orC. The Gag coding sequences were modified to be comparable to codonusage found in highly expressed human genes. In FIG. 11 (Example 1), thepercent A-T content of cDNA sequences corresponding to the mRNA for aknown unstable mRNA and a known stable mRNA are compared to the percentA-T content of native HIV-1SF2 Gag cDNA and to the synthetic Gag cDNAsequence of the present invention. Experiments performed in support ofthe present invention showed that the synthetic Gag sequences werecapable of higher level of protein production (see the Examples)relative to the native Gag sequences. The data in FIG. 11 suggest thatone reason for this increased production is increased stability of themRNA corresponding to the synthetic Gag coding sequences versus the mRNAcorresponding to the native Gag coding sequences.

Second, there are inhibitory (or instability) elements (INS) locatedwithin the coding sequences of the Gag coding sequences (Example 1). TheRRE is a secondary RNA structure that interacts with the HIV encodedRev-protein to overcome the expression down-regulating effects of theINS. To overcome the post-transcriptional activating mechanisms of RREand Rev, the instability elements were inactivated by introducingmultiple point mutations that did not alter the reading frame of theencoded proteins. FIG. 1 shows the original SF2 Gag sequence, thelocation of the INS sequences, and the modifications made to the INSsequences to reduce their effects. The resulting modified codingsequences are presented as a synthetic Gag expression cassette (SEQ IDNO:4).

Modification of the Gag polypeptide coding sequences resulted inimproved expression relative to the wild-type coding sequences in anumber of mammalian cell lines (as well as other types of cell lines,including, but not limited to, insect cells). Further, expression of thesequences resulted in production of virus-like particles (VLPs) by thesecell lines (see below). Similar Gag polypeptide coding sequences can beobtained from a variety of isolates (families, sub-types, strains, etc.)including, but not limited to such other variants include, but are notlimited to, Gag polypeptide encoding sequences obtained from theisolates HIV_(IIIb), HIV_(SF2), HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV),HIV_(LAI), HIV_(MN), HIV-1_(CM235), HIV-1_(US4), other HIV-1 strainsfrom diverse subtypes(e.g., subtypes, A through G, and O), HIV-2 strainsand diverse subtypes (e.g., HIV-2_(UC1) and HIV-2_(UC2)), and simianimmunodeficiency virus (SIV). (See, e.g., Virology, 3rd Edition (W. K.Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D.M. Knipe, eds. 1991; Virology, 3rd Edition (Fields, B N, D M Knipe, P MHowley, Editors, 1996, Lippincott-Raven, Philadelphia, Pa.). Gagpolypeptide encoding sequences derived from these variants can beoptimized and tested for improved expression in mammals by following theteachings of the present specification (see the Examples, in particularExample 1).

2.1.2 Further Modification of Sequences Including HIV-1 Gag Nucleic AcidCoding Sequences

Experiments performed in support of the present invention have shownthat similar modifications of HIV-1 Gag-protease, Gag-reversetranscriptase and Gag-polymerase sequences also result in improvedexpression of the polyproteins, as well as, the production of VLPsformed by polypeptides produced from such modified coding sequences.

For the Gag-protease sequence (wild type, SEQ ID NO:2; modified, SEQ IDNOs:5, 78, 79), the changes in codon usage were restricted to theregions upstream of the −1 frameshift (FIG. 2). Further, inhibitory (orinstability) elements (INS) located within the coding sequences of theGag-protease polypeptide coding sequence were altered as well (indicatedin FIG. 2). Exemplary constructs (which include the −1 frameshift)encoding modified Gag-protease sequences include those shown in SEQ IDNOs:78 and 79 (FIGS. 69 and 70). These are: GP1 (SEQ ID NO:78) in whichthe protease region was also codon optimized and INS inactivated and GP2(SEQ ID NO:79), in which the protease region was only subjected to INSinactivation.

For other Gag-containing sequences, for example the Gag-polymerasesequence (wild type, SEQ ID NO:3; modified, SEQ ID NO:6) or Gag-reversetranscriptase (wild type, SEQ ID NO:77; modified SEQ ID NOs:80-84), thechanges in codon usage are similar to those for the Gag-proteasesequence. Those expression cassettes which contain a frameshift in theGagPol coding sequence are designated “FS(+)” (SEQ ID NOs:80 and 81,FIGS. 71 and 72) while the designation “FS(−)” (SEQ ID Nos: 82, 83 and84, FIGS. 73, 74 and 75) indicates that there is no frameshift utilizedin this coding sequence.

In addition to polyproteins containing HIV-related sequences, thevarious Gag-, Gag-prot, Gag-pol, Gag-reverse transcriptase encodingsequences of the present invention can be fused to other polypeptides(creating chimeric polypeptides) for which an immunogenic response isdesired. An example of such a chimeric protein is the joining of theimproved expression Gag encoding sequences to the Hepatitis C Virus(HCV) core protein. In this case, the HCV-core encoding sequences wereplaced in-frame with the HIV-Gag encoding sequences, resulting in theGag/HCV-core encoding sequence presented as SEQ ID NO:7 (wild typesequence presented as SEQ ID NO:8).

Further sequences useful in the practice of the present inventioninclude, but are not limited to, sequences encoding viralepitopes/antigens {including but not limited to, HCV antigens (e.g., E1,E2; Houghton, M., et al., U.S. Pat. No. 5,714,596, issued Feb. 3, 1998;Houghton, M., et al., U.S. Pat. No. 5,712,088, issued Jan. 27, 1998;Houghton, M., et al., U.S. Pat. No. 5,683,864, issued Nov. 4, 1997;Weiner, A. J., et al., U.S. Pat. No. 5,728,520, issued Mar. 17, 1998;Weiner, A. J., et al., U.S. Pat. No. 5,766,845, issued Jun. 16, 1998;Weiner, A. J., et al., U.S. Pat. No. 5,670,152, issued Sep. 23, 1997;all herein incorporated by reference), HIV antigens (e.g., derived fromnef, tat, rev, vpu, vif, vpr and/or env); and sequences encoding tumorantigens/epitopes. Additional sequences are described below. Also,variations on the orientation of the Gag and other coding sequences,relative to each other, are also described below.

Gag, Gag-protease, Gag-reverse transcriptase and/or Gag-polymerasepolypeptide coding sequences can be obtained from any HIV isolates(different families, subtypes, and strains) including but not limited tothe isolates HIV_(IIIb), HIV_(SF2), HIV_(SF162), HIVus4, HIV_(cm235),HIV_(LAV), HIV_(MN)) (see, e.g., Myers et al. Los Alamos Database, LosAlamos National Laboratory, Los Alamos, N. Mex. (1992); Myers et al.,Human Retroviruses and Aids, 1997, Los Alamos, N. Mex.: Los AlamosNational Laboratory). Synthetic expression cassettes can be generatedusing such coding sequences as starting material by following theteachings of the present specification (e.g., see Example 1). Further,the synthetic expression cassettes of the present invention includerelated Gag polypeptide coding sequences having greater than 75%,preferably greater than 80-85%, more preferably greater than 90-95%, andmost preferably greater than 98% sequence identity (or any integer valuewithin these ranges) to the synthetic expression cassette sequencesdisclosed herein (for example, SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6;and SEQ ID NO:20, the Gag Major Homology Region).

2.1.3 Expression of Synthetic Sequences Encoding HIV-1 Gag and RelatedPolypeptides

Several synthetic Gag-encoding sequences (expression cassettes) of thepresent invention were cloned into a number of different expressionvectors (Example 1) to evaluate levels of expression and production ofVLPs. Two modified synthetic coding sequences are presented as asynthetic Gag expression cassette (SEQ ID NO:4) and a syntheticGag-protease expression cassette (SEQ ID NOs:78 and 79). Other syntheticGag-encoding proteins are presented, for example, as SEQ ID NOs:80through 84. The synthetic DNA fragments for Gag-encoding polypeptides(e.g., Gag, Gag-protease, Gag-polymerase, Gag-reverse transcriptase)were cloned into expression vectors described in Example 1, including, atransient expression vector, CMV-promoter-based mammalian vectors, and ashuttle vector for use in baculovirus expression systems. Correspondingwild-type sequences were cloned into the same vectors.

These vectors were then transfected into a several different cell types,including a variety of mammalian cell lines, (293, RD, COS-7, and CHO,cell lines available, for example, from the A.T.C.C.). The cell lineswere cultured under appropriate conditions and the levels of p24 (Gag)expression in supernatants were evaluated (Example 2). The results ofthese assays demonstrated that expression of synthetic Gag-encodingsequences were significantly higher than corresponding wild-typesequences (Example 2; Table 2).

Further, Western Blot analysis showed that cells containing thesynthetic Gag expression cassette produced the expected 55 kD (p55)protein at higher per-cell concentrations than cells containing thenative expression cassette. The Gag p55 protein was seen in both celllysates and supernatants. The levels of production were significantlyhigher in cell supernatants for cells transfected with the synthetic Gagexpression cassette of the present invention. Experiments performed insupport of the present invention suggest that cells containing thesynthetic Gag-prot expression cassettes produced the expected Gag-protprotein at comparably higher per-cell concentrations than cellscontaining the wild-type expression cassette.

Fractionation of the supernatants from mammalian cells transfected withthe synthetic Gag expression cassette showed that it provides superiorproduction of both p55 protein and VLPs, relative to the wild-type Gagsequences (Examples 6 and 7).

Efficient expression of these Gag-containing polypeptides in mammaliancell lines provides the following benefits: the Gag polypeptides arefree of baculovirus contaminants; production by established methodsapproved by the FDA; increased purity; greater yields (relative tonative coding sequences); and a novel method of producing theGag-containing polypeptides in CHO or other mammalian cells which is notfeasible in the absence of the increased expression obtained using theconstructs of the present invention. Exemplary Mammalian cell linesinclude, but are not limited to, BHK, VERO, HT1080, 293, 293T, RD,COS-7, CHO, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT-4, H9, PM1,CEM, myeloma cells (e.g., SB20 cells) and CEMX174, such cell lines areavailable, for example, from the A.T.C.C.).

A synthetic Gag expression cassette of the present invention alsodemonstrated high levels of expression and VLP production whentransfected into insect cells (Example 7). Further, in addition to ahigher total protein yield, the final product from the syntheticp55-expressed Gag consistently contained lower amounts of contaminatingbaculovirus proteins than the final purified product from the nativep55-expressed Gag.

Further, synthetic Gag expression cassettes of the present inventionhave also been introduced into yeast vectors which were transformed intoand efficiently expressed by yeast cells (Saccharomyces cerevisea; usingvectors as described in Rosenberg, S. and Tekamp-Olson, P., U.S. Pat.No. RE35,749, issued, Mar. 17, 1998, herein incorporated by reference).

In addition to the mammalian and insect vectors described in theExamples, the synthetic expression cassettes of the present inventioncan be incorporated into a variety of expression vectors using selectedexpression control elements. Appropriate vectors and control elementsfor any given cell type can be selected by one having ordinary skill inthe art in view of the teachings of the present specification andinformation known in the art about expression vectors.

For example, a synthetic Gag expression cassette can be inserted into avector which includes control elements operably linked to the desiredcoding sequence, which allow for the expression of the gene in aselected cell-type. For example, typical promoters for mammalian cellexpression include the SV40 early promoter, a CMV promoter such as theCMV immediate early promoter (a CMV promoter can include intron A), RSV,HIV-LTR, the mouse mammary tumor virus LTR promoter (MMLV-LTR), FIV-LTR,the adenovirus major late promoter (Ad MLP), and the herpes simplexvirus promoter, among others. Other nonviral promoters, such as apromoter derived from the murine metallothionein gene, will also finduse for mammalian expression. Typically, transcription termination andpolyadenylation sequences will also be present, located 3′ to thetranslation stop codon. Preferably, a sequence for optimization ofinitiation of translation, located 5′ to the coding sequence, is alsopresent. Examples of transcription terminator/polyadenylation signalsinclude those derived from SV40, as described in Sambrook, et al.,supra, as well as a bovine growth hormone terminator sequence. Introns,containing splice donor and acceptor sites, may also be designed intothe constructs for use with the present invention (Chapman et al., Nuc.Acids Res. (1991) 19:3979-3986).

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence (Chapman at al., Nuc. Acids Res.(1991) 19:3979-3986).

The desired synthetic Gag polypeptide encoding sequences can be clonedinto any number of commercially available vectors to generate expressionof the polypeptide in an appropriate host system. These systems include,but are not limited to, the following: baculovirus expression {Reilly,P. R., et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL(1992); Beames, et al., Biotechniques 11:378 (1991); Pharmingen;Clontech, Palo Alto, Calif.)), vaccinia expression {Earl, P. L., et al.,“Expression of proteins in mammalian cells using vaccinia” In CurrentProtocols in Molecular Biology (F. M. Ausubel, et al. Eds.), GreenePublishing Associates & Wiley Interscience, New York (1991); Moss, B.,et al., U.S. Pat. No. 5,135,855, issued 4 Aug. 1992}, expression inbacteria {Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley and Sons, Inc., Media PA; Clontech}, expression inyeast {Rosenberg, S. and Tekamp-Olson, P., U.S. Pat. No. RE35,749,issued, Mar. 17, 1998, herein incorporated by reference; Shuster, J. R.,U.S. Pat. No. 5,629,203, issued May 13, 1997, herein incorporated byreference; Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(1-2):79-93(1992); Romanos, M. A., at al., Yeast 8(6):423-488 (1992); Goeddel, D.V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R. Fink,Methods in Enzymology 194 (1991)}, expression in mammalian cells{Clontech; Gibco-BRL, Ground Island, N. Y.; e.g., Chinese hamster ovary(CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983);1983, Lau, Y. F., et al., Mol. Cell. Biol. 4:1469-1475 (1984); Kaufman,R. J., “Selection and coamplification of heterologous genes in mammaliancells,” in Methods in Enzymology, vol. 185, pp 537-566. Academic Press,Inc., San Diego Calif. (1991)}, and expression in plant cells {plantcloning vectors, Clontech Laboratories, Inc., Palo Alto, Calif., andPharmacia LKB Biotechnology, Inc., Pistcataway, N.J.; Hood, E., et al.,J. Bacteriol. 168:1291-1301 (1986); Nagel, R., et al., FEMS Microbiol.Lett. 67:325 (1990); An, et al., “Binary Vectors”, and others in PlantMolecular Biology Manual A3:1-19 (1988); Miki, B. L. A., et al., pp.249-265, and others in Plant DNA Infectious Agents (Hohn, T., et al.,eds.) Springer-Verlag, Wien, Austria, (1987); Plant Molecular Biology:Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley,1997; Miglani, Gurbachan Dictionary of Plant Genetics and MolecularBiology, New York, Food Products Press, 1998; Henry, R. J., PracticalApplications of Plant Molecular Biology, New York, Chapman & Hall,1997}.

Also included in the invention is an expression vector, such as the CMVpromoter-containing vectors described in Example 1, containing codingsequences and expression control elements which allow expression of thecoding regions in a suitable host. The control elements generallyinclude a promoter, translation initiation codon, and translation andtranscription termination sequences, and an insertion site forintroducing the insert into the vector. Translational control elementshave been reviewed by M. Kozak (e.g., Kozak, M., Mamm. Genome7(8):563-574, 1996; Kozak, M., Biochimie 76(9):815-821, 1994; Kozak, M.,J Cell Biol 108(2):229-241, 1989; Kozak, M., and Shatkin, A. J., MethodsEnzymol 60:360-375, 1979).

Expression in yeast systems has the advantage of commercial production.Recombinant protein production by vaccinia and CHO cell line have theadvantage of being mammalian expression systems. Further, vaccinia virusexpression has several advantages including the following: (i) its widehost range; (ii) faithful post-transcriptional modification, processing,folding, transport, secretion, and assembly of recombinant proteins;(iii) high level expression of relatively soluble recombinant proteins;and (iv) a large capacity to accommodate foreign DNA.

The recombinantly expressed polypeptides from synthetic Gag-encodingexpression cassettes are typically isolated from lysed cells or culturemedia. Purification can be carried out by methods known in the artincluding salt fractionation, ion exchange chromatography, gelfiltration, size-exclusion chromatography, size-fractionation, andaffinity chromatography. Immunoaffinity chromatography can be employedusing antibodies generated based on, for example, Gag antigens.

Advantages of expressing the Gag-containing proteins of the presentinvention using mammalian cells include, but are not limited to, thefollowing: well-established protocols for scale-up production; theability to produce VLPs; cell lines are suitable to meet goodmanufacturing process (GMP) standards; culture conditions for mammaliancells are known in the art.

2.1.4 Modification of HIV-1 Env Nucleic Acid Coding Sequences

One aspect of the present invention is the generation of HIV-1 Envprotein coding sequences, and related sequences, having improvedexpression relative to the corresponding wild-type sequence. Exemplaryembodiments of the present invention are illustrated herein modifyingthe Env protein wild-type sequences obtained from the HIV-1 subtype Bstrains HIV-1US4 and HIV-1SF162 (Myers et al., Los Alamos Database, LosAlamos National Laboratory, Los Alamos, N. Mex. (1992); Myers et al.,Human Retroviruses and Aids, 1997, Los Alamos, N. Mex.: Los AlamosNational Laboratory). Env sequence obtained from other HIV variants maybe manipulated in similar fashion following the teachings of the presentspecification. Such other variants include those described above inSection 2.1.1 and on the World Wide Web (Internet), for example athttp://hiv-web.lan1.gov/cgi-bin/hivDB3/public/wdb/ssampublic andhttp://hiv-web.lan1.gov.

First, the HIV-1 codon usage pattern was modified so that the resultingnucleic acid coding sequence was comparable to codon usage found inhighly expressed human genes (Example 1). The HIV codon usage reflects ahigh content of the nucleotides A or T of the codon-triplet. The effectof the HIV-1 codon usage is a high AT content in the DNA sequence thatresults in a decreased translation ability and instability of the mRNA.In comparison, highly expressed human codons prefer the nucleotides G orC. The Env coding sequences were modified to be comparable to codonusage found in highly expressed human genes. Experiments performed insupport of the present invention showed that the synthetic Env sequenceswere capable of higher level of protein production (see the Examples)relative to the native Env sequences. One reason for this increasedproduction may be increased stability of the mRNA corresponding to thesynthetic Env coding sequences versus the mRNA corresponding to thenative Env coding sequences.

Modification of the Env polypeptide coding sequences resulted inimproved expression relative to the wild-type coding sequences in anumber of mammalian cell lines. Similar Env polypeptide coding sequencescan be obtained from a variety of isolates (families, sub-types, etc.).Env polypeptide encoding sequences derived from these variants can beoptimized and tested for improved expression in mammals by following theteachings of the present specification (see the Examples, in particularExample 2).

2.1.5 Further Modification of HIV-1 Env Nucleic Acid Coding Sequences

In addition to proteins containing HIV-related sequences, the Envencoding sequences of the present invention can be fused to otherpolypeptides (creating chimeric polypeptides). Also, variations on theorientation of the Env and other coding sequences, relative to eachother, are contemplated. Further, the HIV protein encoding cassettes ofthe present invention can be co-expressed using one vector or multiplevectors. In addition, the polyproteins can be operably linked to thesame or different promoters.

Env polypeptide coding sequences can be obtained from any HIV isolates(different families, subtypes, and strains) including but not limited tothe isolates HIV_(IIIb), HIV_(SF2), HIV_(us4), HIV_(CM235), HIV_(SF162),HIV_(LAV), HIV_(LAI), HIV_(MN)) (see, e.g., Myers et al., Los AlamosDatabase, Los Alamos National Laboratory, Los Alamos, N. Mex. (1992);Myers et al., Human Retroviruses and Aids, 1997, Los Alamos, N. Mex.:Los Alamos National Laboratory). Synthetic expression cassettes can begenerated using such coding sequences as starting material by followingthe teachings of the present specification (e.g., see Example 1).Further, the synthetic expression cassettes (and purifiedpolynucleotides) of the present invention include related Envpolypeptide coding sequences having greater than 90%, preferably greaterthan 92%, more preferably greater than 95%, and most preferably greaterthan 98% sequence identity to the synthetic expression cassettesequences disclosed herein (for example, SEQ ID NOs:71-72; and/or thesequences presented in Tables 1A and 1B) when the sequences of thepresent invention are used as the query sequence.

2.1.6 Expression of Synthetic Sequences Encoding HIV-1 Env and RelatedPolypeptides

Several synthetic Env-encoding sequences (expression cassettes) of thepresent invention were cloned into a number of different expressionvectors (Example 1) to evaluate levels of expression and production ofEnv polypeptide. A modified synthetic coding sequence is presented assynthetic Env expression cassettes (Example 1, e.g., Tables 1A and 1B).The synthetic DNA fragments for Env were cloned into eucaryoticexpression vectors described in Example 1 and in Section 2.1.3 above,including, a transient expression vector and CMV-promoter-basedmammalian vectors. Corresponding wild-type sequences were cloned intothe same vectors.

These vectors were then transfected into a several different cell types,including a variety of mammalian cell lines, (293, RD, COS-7, and CHO,cell lines available, for example, from the A.T.C.C.). The cell lineswere cultured under appropriate conditions and the levels of gp120,gp140 and gp160 Env expression in supernatants were evaluated (Example2). Env polypeptides include, but are not limited to, for example,native gp160, oligomeric gp140, monomeric gp120 as well as modifiedsequences of these polypeptides. The results of these assaysdemonstrated that expression of synthetic Env encoding sequences weresignificantly higher than corresponding wild-type sequences (Example 2;Tables 3 and 4).

Further, Western Blot analysis showed that cells containing thesynthetic Env expression cassette produced the expected protein (gp120,gp140 or gp160) at higher per-cell concentrations than cells containingthe native expression cassette. The Env proteins were seen in both celllysates and supernatants. The levels of production were significantlyhigher in cell supernatants for cells transfected with the synthetic Envexpression cassettes of the present invention as compared to wild type.

Fractionation of the supernatants from mammalian cells transfected withthe synthetic Env expression cassettes showed that it provides superiorproduction of Env proteins, relative to the wild-type Env sequences(Examples 2 and 3).

Efficient expression of these Env-containing polypeptides in mammaliancell lines provides the following benefits: the Env polypeptides arefree of baculovirus or other viral contaminants; production byestablished methods approved by the FDA; increased purity; greateryields (relative to native coding sequences); and a novel method ofproducing the Env-containing polypeptides in CHO cells which is lessfeasible in the absence of the increased expression obtained using theconstructs of the present invention.

Exemplary cell lines (e.g., mammalian, yeast, insect, etc.) includethose described above in Section 2.1.3 for Gag-containing constructs.Further, appropriate vectors and control elements (e.g., promoters,enhancers, polyadenylation sequences, etc.) for any given cell type canbe selected, as described above in Section 2.1.3, by one having ordinaryskill in the art in view of the teachings of the present specificationand information known in the art about expression vectors. In addition,the recombinantly expressed polypeptides from synthetic Env-encodingexpression cassettes are typically isolated and purified from lysedcells or culture media, as described above for Gag-encoding expressioncassettes. An exemplary purification is described in Example 4 and shownin FIG. 60.

2.1.7 Modification of HIV-1 Tat Nucleic Acid Coding Sequences

Another aspect of the present invention is the generation of HIV-1 tatprotein coding sequences, and related sequences, having improvedexpression relative to the corresponding wild-type sequence. Exemplaryembodiments of the present invention are illustrated herein modifyingthe tat wild-type nucleotide sequence (SEQ ID NO:85, FIG. 76) obtainedfrom SF162 as described above. Exemplary synthetic tat constructs areshown in SEQ ID NO:87, which depicts a tat construct encoding afull-length tat polypeptide from strain SF162; SEQ ID NO:88, whichdepicts a tat construct encoding a tat polypeptide having the cysteinresidue at position 22 changed; and SEQ ID NO:89, which depicts a tatconstruct encoding the amino terminal portion of a tat polypeptide fromstrain SF162. The amino portion of the tat protein appears to containmany of the epitopes that induce an immune response. In addition,further modifications include replacement or deletion of the cysteinresidue at position 22, for example with a valine residue, an alanineresidue or a glycine residue (SEQ ID Nos: 88 and 89, FIGS. 79 and 81),see, e.g., Caputo et al. (1996) Gene Ther. 3:235. In FIG. 81, whichdepicts a tat construct encoding the amino terminal portion of a tatpolypeptide, the nucleotides (nucleotides 64-66) encoding the cysteinresidues are underlined. The design and construction of suitableconstruct can be readily done using the teachings of the presentspecification. As with Gag, pol, prot and Env, tat polypeptide codingsequences can be obtained from a variety of isolates (families,sub-types, etc.).

Modification of the tat polypeptide coding sequences result in improvedexpression relative to the wild-type coding sequences in a number ofcell lines (e.g., mammalian, yeast, bacterial and insect cells). Tatpolypeptide encoding sequences derived from these variants can beoptimized and tested for improved expression in mammals by following theteachings of the present specification (see the Examples, in particularExample 2).

Various forms of the different embodiments of the invention, describedherein, may be combined. For example, polynucleotides may be derivedfrom the polynucleotide sequences of the present invention, including,but not limited to, coding sequences for Gag polypeptides, Envpolypeptides, polymerase polypeptides, protease polypeptides, tatpolypeptides, and reverse transcriptase polypeptides. Further, thepolynucleotide coding sequences of the present invention may be combinedinto multi-cistronic expression cassettes where typically each codingsequence for each polypeptide is preceded by IRES sequences.

2.2 Production of Virus-Like Particles and Use of the Constructs of thePresent Invention to Create Packaging Cell Lines

The group-specific antigens (Gag) of human immunodeficiency virus type-1(HIV-1) self-assemble into noninfectious virus-like particles (VLP) thatare released from various eucaryotic cells by budding (reviewed byFreed, E. O., Virology 251:1-15, 1998). The synthetic expressioncassettes of the present invention provide efficient means for theproduction of HIV-Gag virus-like particles (VLPs) using a variety ofdifferent cell types, including, but not limited to, mammalian cells.

Viral particles can be used as a matrix for the proper presentation ofan antigen entrapped or associated therewith to the immune system of thehost. For example, U.S. Pat. No. 4,722,840 describes hybrid particlescomprised of a particle-forming fragment of a structural protein from avirus, such as a particle-forming fragment of hepatitis B virus (HBV)surface antigen (HBsAg), fused to a heterologous polypeptide. Tindle etal., Virology (1994) 200:547-557, describes the production and use ofchimeric HBV core antigen particles containing epitopes of humanpapillomavirus (HPV) type 16 E7 transforming protein.

Adams et al., Nature (1987) 329:68-70, describes the recombinantproduction of hybrid HIVgp120:Ty VLPs in yeast and Brown et al.,Virology (1994) 198:477-488, the production of chimeric proteinsconsisting of the VP2 protein of human parvovirus B19 and epitopes fromhuman herpes simplex virus type 1, as well as mouse hepatitis virus A59.Wagner et al., (Virology (1994) 200:162-175, Brand et al., J. Virol.Meth. (1995) 51:153-168; Virology (1996) 220:128-140) and Wolf, et al.,(EP 0 449 116 A1, published 2 Oct. 1991; WO 96/30523, published 3 Oct.1996) describe the assembly of chimeric HIV-1 p55Gag particles. U.S.Pat. No. 5,503,833 describes the use of rotavirus VP6 spheres forencapsulating and delivering therapeutic agents.

2.2.1 VLP Production Using the Synthetic Expression Cassettes of thePresent Invention

Experiments performed in support of the present invention havedemonstrated that the synthetic expression cassettes of the presentinvention provide superior production of both protein and VLPs, relativeto native coding sequences (Examples 7 and 15). Further, electronmicroscopic evaluation of VLP production (Examples 6 and 15, FIGS. 3A-Band 65A-F) showed that free and budding immature virus particles of theexpected size were produced by cells containing the synthetic expressioncassettes.

Using the synthetic expression cassettes of the present invention,rather than native coding sequences, for the production of virus-likeparticles provide several advantages. First, VLPs can be produced inenhanced quantity making isolation and purification of the VLPs easier.Second, VLPs can be produced in a variety of cell types using thesynthetic expression cassettes, in particular, mammalian cell lines canbe used for VLP production, for example, CHO cells. Production using CHOcells provides (i) VLP formation; (ii) correct myristylation andbudding; (iii) absence of non-mammalian cell contaminants (e.g., insectviruses and/or cells); and (iv) ease of purification. The syntheticexpression cassettes of the present invention are also useful forenhanced expression in cell-types other than mammalian cell lines. Forexample, infection of insect cells with baculovirus vectors encoding thesynthetic expression cassettes resulted in higher levels of totalprotein yield and higher levels of VLP production (relative to wild-typecoding sequences). Further, the final product from insect cells infectedwith the baculovirus-Gag synthetic expression cassettes consistentlycontained lower amounts of contaminating insect proteins than the finalproduct when wild-type coding sequences were used (Examples).

VLPs can spontaneously form when the particle-forming polypeptide ofinterest is recombinantly expressed in an appropriate host cell. Thus,the VLPs produced using the synthetic expression cassettes of thepresent invention are conveniently prepared using recombinanttechniques. As discussed below, the Gag polypeptide encoding syntheticexpression cassettes of the present invention can include otherpolypeptide coding sequences of interest (for example, Env, tat, rev,HIV protease, HIV polymerase, HCV core; see, Example 1). Expression ofsuch synthetic expression cassettes yields VLPs comprising the productof the synthetic expression cassette, as well as, the polypeptide ofinterest.

Once coding sequences for the desired particle-forming polypeptides havebeen isolated or synthesized, they can be cloned into any suitablevector or replicon for expression. Numerous cloning vectors are known tothose of skill in the art, and the selection of an appropriate cloningvector is a matter of choice. See, generally, Ausubel et al, supra orSambrook et al, supra. The vector is then used to transform anappropriate host cell. Suitable recombinant expression systems include,but are not limited to, bacterial, mammalian, baculovirus/insect,vaccinia, Semliki Forest virus (SFV), Alphaviruses (such as, Sindbis,Venezuelan Equine Encephalitis (VEE)), mammalian, yeast and Xenopusexpression systems, well known in the art. Particularly preferredexpression systems are mammalian cell lines, vaccinia, Sindbis, insectand yeast systems.

For example, a number of mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (A.T.C.C.), such as, but not limited to, Chinese hamsterovary (CHO) cells, 293 cells, HeLa cells, baby hamster kidney (BHK)cells, mouse myeloma (SB20), monkey kidney cells (COS), as well asothers. Similarly, bacterial hosts such as E. coli, Bacillus subtilis,and Streptococcus spp., will find use with the present expressionconstructs. Yeast hosts useful in the present invention include interalia, Saccharomyces cerevisiae, Candida albicans, Candida maltose,Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis,Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe andYarrowia lipolytica. Insect cells for use with baculovirus expressionvectors include, inter alia, Aedes aegypti, Autographa californica ,Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, andTrichoplusia ni. See, e.g., Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987). Fungal hosts include, forexample, Aspergillus.

Viral vectors can be used for the production of particles in eucaryoticcells, such as those derived from the pox family of viruses, includingvaccinia virus and avian poxvirus. Additionally, a vaccinia basedinfection/transfection system, as described in Tomei et al., J. Virol.(1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993)74:1103-1113, will also find use with the present invention. In thissystem, cells are first infected in vitro with a vaccinia virusrecombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the DNA of interest, driven by a T7 promoter. Thepolymerase expressed in the cytoplasm from the vaccinia virusrecombinant transcribes the transfected DNA into RNA which is thentranslated into protein by the host translational machinery.Alternately, T7 can be added as a purified protein or enzyme as in the“Progenitor” system (Studier and Moffatt, J. Mol. Biol. (1986)189:113-130). The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation product(s).

Depending on the expression system and host selected, the VLPS areproduced by growing host cells transformed by an expression vector underconditions whereby the particle-forming polypeptide is expressed andVLPs can be formed. The selection of the appropriate growth conditionsis within the skill of the art. If the VLPs are formed intracellularly,the cells are then disrupted, using chemical, physical or mechanicalmeans, which lyse the cells yet keep the VLPs substantially intact. Suchmethods are known to those of skill in the art and are described in,e.g., Protein Purification Applications: A Practical Approach, (E. L. V.Harris and S. Angal, Eds., 1990).

The particles are then isolated (or substantially purified) usingmethods that preserve the integrity thereof, such as, by densitygradient centrifugation, e.g., sucrose gradients, PEG-precipitation,pelleting, and the like (see, e.g., Kirnbauer et al. J. Virol. (1993)67:6929-6936), as well as standard purification techniques including,e.g., ion exchange and gel filtration chromatography.

VLPs produced by cells containing the synthetic expression cassettes ofthe present invention can be used to elicit an immune response whenadministered to a subject. One advantage of the present invention isthat VLPs can be produced by mammalian cells carrying the syntheticexpression cassettes at levels previously not possible. As discussedabove, the VLPs can comprise a variety of antigens in addition to theGag polypeptides (e.g., Env, tat, Gag-protease, Gag-polymerase,Gag-HCV-core). Purified VLPs, produced using the synthetic expressioncassettes of the present invention, can be administered to a vertebratesubject, usually in the form of vaccine compositions. Combinationvaccines may also be used, where such vaccines contain, for example,other subunit proteins derived from HIV or other organisms (e.g., env)or gene delivery vaccines encoding such antigens. Administration cantake place using the VLPs formulated alone or formulated with otherantigens. Further, the VLPs can be administered prior to, concurrentwith, or subsequent to, delivery of the synthetic expression cassettesfor DNA immunization (see below) and/or delivery of other vaccines.Also, the site of VLP administration may be the same or different asother vaccine compositions that are being administered. Gene deliverycan be accomplished by a number of methods including, but are notlimited to, immunization with DNA, alphavirus vectors, pox virusvectors, and vaccinia virus vectors.

VLP immune-stimulating (or vaccine) compositions can include variousexcipients, adjuvants, carriers, auxiliary substances, modulatingagents, and the like. The immune stimulating compositions will includean amount of the VLP/antigen sufficient to mount an immunologicalresponse. An appropriate effective amount can be determined by one ofskill in the art. Such an amount will fall in a relatively broad rangethat can be determined through routine trials and will generally be anamount on the order of about 0.1 μg to about 1000 μg, more preferablyabout 1 μg to about 300 μg, of VLP/antigen.

A carrier is optionally present which is a molecule that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycollic acids, polymeric amino acids, amino acidcopolymers, lipid aggregates (such as oil droplets or liposomes), andinactive virus particles. Examples of particulate carriers include thosederived from polymethyl methacrylate polymers, as well as microparticlesderived from poly(lactides) and poly(lactide-co-glycolides), known asPLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee JP, et al., J. Microencapsul. 14(2):197-210, 1997; O'Hagan D T, et al.,Vaccine 11(2):149-54, 1993. Such carriers are well known to those ofordinary skill in the art. Additionally, these carriers may function asimmunostimulating agents (“adjuvants”). Furthermore, the antigen may beconjugated to a bacterial toxoid, such as toxoid from diphtheria,tetanus, cholera, etc., as well as toxins derived from E. coli.

Such adjuvants include, but are not limited to: (1) aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) oil-in-water emulsion formulations (with or withoutother specific immunostimulating agents such as muramyl peptides (seebelow) or bacterial cell wall components), such as for example (a) MF59(International Publication No. WO 90/14837), containing 5% Squalene,0.5% Tween 80, and 0.5% Span 85 (optionally containing various amountsof MTP-PE (see below), although not required) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below)either microfluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponinadjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.)may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), beta chemokines (MIP, 1-alpha, 1-beta Rantes,etc.); (6) detoxified mutants of a bacterial ADP-ribosylating toxin suchas a cholera toxin (CT), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (see, e.g.,International Publication Nos. WO93/13202 and WO92/19265); and (7) othersubstances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Dosage treatment with the VLP composition may be a single dose scheduleor a multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals, chosen to maintainand/or reinforce the immune response, for example at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by thepotency of the modality, the vaccine delivery employed, the need of thesubject and be dependent on the judgment of the practitioner.

If prevention of disease is desired (e.g., reduction of symptoms,recurrences or of disease progression), the antigen carrying VLPs aregenerally administered prior to primary infection with the pathogen ofinterest. If treatment is desired, e.g., the reduction of symptoms orrecurrences, the VLP compositions are generally administered subsequentto primary infection.

2.2.2 Using the Synthetic Expression Cassettes of the Present Inventionto Create Packaging Cell Lines

A number of viral based systems have been developed for use as genetransfer vectors for mammalian host cells. For example, retroviruses (inparticular, lentiviral vectors) provide a convenient platform for genedelivery systems. A coding sequence of interest (for example, a sequenceuseful for gene therapy applications) can be inserted into a genedelivery vector and packaged in retroviral particles using techniquesknown in the art. Recombinant virus can then be isolated and deliveredto cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described, including, for example, thefollowing: (U.S. Pat. No. 5,219,740; Miller et al. (1989) Biotechniques7:980; Miller, A. D. (1990) Human Gene Therapy 1:5; Scarpa et al. (1991)Virology 180:849; Burns et al. (1993) Proc. Natl. Acad. Sci. USA90:8033; Boris-Lawrie et al. (1993) Cur. Opin. Genet. Develop. 3:102; GB2200651; EP 0415731; EP 0345242; WO 89/02468; WO 89/05349; WO 89/09271;WO 90/02806; WO 90/07936; WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; WO 93/11230; WO 93/10218; WO 91/02805; in U.S. Pat. No.5,219,740; U.S. Pat. No. 4,405,712; U.S. Pat. No. 4,861,719; U.S. Pat.No. 4,980,289 and U.S. Pat. No. 4,777,127; in U.S. Ser. No. 07/800,921;and in Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res53:962-967; Ram (1993) Cancer Res 53:83-88; Takamiya (1992) Neurosci Res33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153;Cane (1984) Proc Natl Acad Sci USA 81; 6349; and Miller (1990) HumanGene Therapy 1.

Sequences useful for gene therapy applications include, but are notlimited to, the following. Factor VIII cDNA, including derivatives anddeletions thereof (International Publication Nos. WO 96/21035, WO97/03193, WO 97/03194, WO 97/03195, and WO 97/03191, all of which arehereby incorporated by reference). Factor IX cDNA (Kurachi et al. (1982)Proc. Natl. Acad. Sci. USA 79:6461-6464). Factor V cDNA can be obtainedfrom pMT2-V (Jenny (1987) Proc. Natl. Acad. Sci. USA 84:4846, A.T.C.C.Deposit No. 40515). A full-length factor V cDNA, or a B domain deletionor B domain substitution thereof, can be used. B domain deletions offactor V, include those reported by Marquette (1995) Blood 86:3026 andKane (1990) Biochemistry 29:6762. Antithrombin III cDNA (Prochownik(1983) J. Biol. Chem. 258:8389, A.T.C.C. Deposit No. 57224/57225).Protein C encoding cDNA (Foster (1984) Proc. Natl. Acad. Sci. USA81:4766; Beckmann (1985) Nucleic Acids Res. 13:5233). Prothrombin cDNAcan be obtained by restriction enzyme digestion of a published vector(Degen (1983) Biochemistry 22:2087). The endothelial cell surfaceprotein, thrombomodulin, is a necessary cofactor for the normalactivation of protein C by thrombin. A soluble recombinant form has beendescribed (Parkinson (1990) J. Biol. Chem. 265:12602; Jackman (1987)Proc. Natl. Acad. Sci. USA 84:6425; Shirai (1988) J. Biochem. 103:281;Wen (1987) Biochemistry 26:4350; Suzuki (1987) EMBO J. 6:1891, A.T.C.C.Deposit No. 61348, 61349).

Many genetic diseases caused by inheritance of defective genes result inthe failure to produce normal gene products, for example, thalassemia,phenylketonuria, Lesch-Nyhan syndrome, severe combined immunodeficiency(SCID), hemophilia A and B, cystic fibrosis, Duchenne's MuscularDystrophy, inherited emphysema and familial hypercholesterolemia(Mulligan et al. (1993) Science 260:926; Anderson et al. (1992) Science256:808; Friedman et al. (1989) Science 244:1275). Although geneticdiseases may result in the absence of a gene product, endocrinedisorders, such as diabetes and hypopituitarism, are caused by theinability of the gene to produce adequate levels of the appropriatehormone insulin and human growth hormone respectively.

In one aspect, gene therapy employing the constructs and methods of thepresent invention involves the introduction of normal recombinant genesinto T cells so that new or missing proteins are produced by the T cellsafter introduction or reintroduction thereof into a patient. A number ofgenetic diseases have been selected for treatment with gene therapy,including adenine deaminase deficiency, cystic fibrosis, α₁-antitrypsindeficiency, Gaucher's syndrome, as well as non-genetic diseases.

In particular, Gaucher's syndrome is a genetic disorder characterized bya deficiency of the enzyme glucocerebrosidase. This enzyme deficiencyleads to the accumulation of glucocerebroside in the lysosomes of allcells in the body. For a review see Science 256:794 (1992) and Scriveret al., The Metabolic Basis of Inherited Disease, 6th ed., vol. 2, page1677). Thus, gene transfer vectors that express glucocerebrosidase canbe constructed for use in the treatment of this disorder. Likewise, genetransfer vectors encoding lactase can be used in the treatment ofhereditary lactose intolerance, those expressing AD can be used fortreatment of ADA deficiency, and gene transfer vectors encodingα₁-antitrypsin can be used to treat α₁-antitrypsin deficiency. SeeLedley, F. D. (1987) J. Pediatrics 110:157-174, Verma, I. (November1987) Scientific American pp. 68-84, and International Publication No.WO 95/27512 entitled “Gene Therapy Treatment for a Variety of Diseasesand Disorders,” for a description of gene therapy treatment of geneticdiseases.

In still further embodiments of the invention, nucleotide sequenceswhich can be incorporated into a gene transfer vector include, but arenot limited to, proteins associated with enzyme-deficiency disorders,such as the cystic fibrosis transmembrane regulator (see, for example,U.S. Pat. No. 5,240,846 and Larrick et al. (1991) Gene TherapyApplications of Molecular Biology, Elsevier, New York and adenosinedeaminase (ADA) (see U.S. Pat. No. 5,399,346); growth factors, or anagonist or antagonist of a growth factor (Bandara et al. (1992) DNA andCell Biology, 11:227); one or more tumor suppressor genes such as p53,Rb, or C-CAMI (Kleinerman et al. (1995) Cancer Research 55:2831); amolecule that modulates the immune system of an organism, such as a HLAmolecule (Nabel et al. (1993) Proc. Natl. Acad. Sci. USA 90:11307); aribozyme (Larsson et al. (1996) Virology 219:161); a peptide nucleicacid (Hirshman et al. (1996) J. Invest. Med. 44:347); an antisensemolecule (Bordier et al. (1995) Proc. Natl. Acad. Sci. USA 92:9383)which can be used to down-regulate the expression or synthesis ofaberrant or foreign proteins, such as HIV proteins or a wide variety ofoncogenes such as p53 (Hesketh, The Oncogene Facts Book, Academic Press,New York, (1995); a biopharmaceutical agent or antisense molecule usedto treat HIV-infection, such as an inhibitor of p24 (Nakashima et al.(1994) Nucleic Acids Res. 22:5004); or reverse-transcriptase (see,Bordier, supra).

Other proteins of therapeutic interest can be expressed in vivo by genetransfer vectors using the methods of the invention. For instancesustained in vivo expression of tissue factor inhibitory protein (TFPI)is useful for treatment of conditions including sepsis and DIC and inpreventing reperfusion injury. (See International Publications Nos. WO93/24143, WO 93/25230 and WO 96/06637). Nucleic acid sequences encodingvarious forms of TFPI can be obtained, for example, as described in U.S.Pat. Nos. 4,966,852; 5,106,833; and 5,466,783, and incorporated into thegene transfer vectors described herein.

Erythropoietin (EPO) and leptin can also be expressed in vivo fromgenetically modified T cells according to the methods of the invention.For instance EPO is useful in gene therapy treatment of a variety ofdisorders including anemia (see International Publication No. WO95/13376 entitled “Gene Therapy for Treatment of Anemia”). Sustaineddelivery of leptin by the methods of the invention is useful intreatment of obesity. See International Publication No. WO 96/05309 fora description of the leptin gene and the use thereof in the treatment ofobesity.

A variety of other disorders can also be treated by the methods of theinvention. For example, sustained in vivo systemic production ofapolipoprotein E or apolipoprotein A from genetically modified T cellscan be used for treatment of hyperlipidemia (see Breslow et al. (1994)Biotechnology 12:365). Sustained production of angiotensin receptorinhibitor (Goodfriend et al. (1996) N. Engl. J. Med. 334:1469) can beprovided by the methods described herein. As yet an additional example,the long term in vivo systemic production of angiostatin is useful inthe treatment of a variety of tumors. (See O'Reilly et al. (1996) NatureMed. 2:689).

In other embodiments, gene transfer vectors can be constructed to encodea cytokine or other immunomodulatory molecule. For example, nucleic acidsequences encoding native IL-2 and gamma-interferon can be obtained asdescribed in U.S. Pat. Nos. 4,738,927 and 5,326,859, respectively, whileuseful muteins of these proteins can be obtained as described in U.S.Pat. No. 4,853,332. Nucleic acid sequences encoding the short and longforms of mCSF can be obtained as described in U.S. Pat. Nos. 4,847,201and 4,879,227, respectively. In particular aspects of the invention,retroviral vectors expressing cytokine or immunomodulatory genes can beproduced as described herein (for example, employing the packaging celllines of the present invention) and in International Application No. PCTUS 94/02951, entitled “Compositions and Methods for CancerImmunotherapy.”

Examples of suitable immunomodulatory molecules for use herein includethe following: IL-1 and IL-2 (Karupiah et al. (1990) J. Immunology144:290-298, Weber et al. (1987). J. Exp. Med. 166:1716-1733, Gansbacheret al. (1990) J. Exp. Med. 172:1217-1224, and U.S. Pat. No. 4,738,927);IL-3 and IL-4 (Tepper et al. (1989) Cell 57:503-512, Golumbek et al.(1991) Science 254:713-716, and U.S. Pat. No. 5,017,691); IL-5 and IL-6(Brakenhof et al. (1987) J. Immunol. 139:4116-4121, and InternationalPublication No. WO 90/06370); IL-7 (U.S. Pat. No. 4,965,195); IL-8,IL-9, IL-10, IL-11, IL-12, and IL-13 (Cytokine Bulletin, Summer 1994);IL-14 and IL-15; alpha interferon (Finter et al. (1991) Drugs42:749-765, U.S. Pat. Nos. 4,892,743 and 4,966,843, InternationalPublication No. WO 85/02862, Nagata et al. (1980) Nature 284:316-320,Familletti et al. (1981) Methods in Enz. 78:387-394, Twu et al. (1989)Proc. Natl. Acad. Sci. USA 86:2046-2050, and Faktor et al. (1990)Oncogene 5:867-872); beta-interferon (Seif et al. (1991) J. Virol.65:664-671); gamma-interferons (Radford et al. (1991) The AmericanSociety of Hepatology 20082015, Watanabe et al. (1989) Proc. Natl. Acad.Sci. USA 86:9456-9460, Gansbacher et al. (1990) Cancer Research50:7820-7825, Maio et al. (1989) Can. Immunol. Immunother. 30:34-42, andU.S. Pat. Nos. 4,762,791 and 4,727,138); G-CSF (U.S. Pat. Nos. 4,999,291and 4,810,643); GM-CSF (International Publication No. WO 85/04188);tumor necrosis factors (TNFs) (Jayaraman et al. (1990) J. Immunology144:942-951); CD3 (Krissanen et al. (1987) Immunogenetics 26:258-266);ICAM-1 (Altman et al. (1989) Nature 338:512-514, Simmons et al. (1988)Nature 331:624-627); ICAM-2, LFA-1, LFA-3 (Wallner et al. (1987) J. Exp.Med. 166:923-932); MHC class I molecules, MHC class II molecules,B7.1-.3, β₂-microglobulin (Parnes et al. (1981) Proc. Natl. Acad. Sci.USA 78:2253-2257); chaperones such as calnexin; and MHC-linkedtransporter proteins or analogs thereof (Powis et al. (1991) Nature354:528-531). Immunomodulatory factors may also be agonists,antagonists, or ligands for these molecules. For example, soluble formsof receptors can often behave as antagonists for these types of factors,as can mutated forms of the factors themselves.

Nucleic acid molecules that encode the above-described substances, aswell as other nucleic acid molecules that are advantageous for usewithin the present invention, may be readily obtained from a variety ofsources, including, for example, depositories such as the American TypeCulture Collection, or from commercial sources such as BritishBio-Technology Limited (Cowley, Oxford England). Representative examplesinclude BBG 12 (containing the GM-CSF gene coding for the mature proteinof 127 amino acids), BBG 6 (which contains sequences encoding gammainterferon), A.T.C.C. Deposit No. 39656 (which contains sequencesencoding TNF), A.T.C.C. Deposit No. 20663 (which contains sequencesencoding alpha-interferon), A.T.C.C. Deposit Nos. 31902, 31902 and 39517(which contain sequences encoding beta-interferon), A.T.C.C. Deposit No.67024 (which contains a sequence which encodes Interleukin-1b), A.T.C.C.Deposit Nos. 39405, 39452, 39516, 39626 and 39673 (which containsequences encoding Interleukin-2), A.T.C.C. Deposit Nos. 59399, 59398,and 67326 (which contain sequences encoding Interleukin-3), A.T.C.C.Deposit No. 57592 (which contains sequences encoding Interleukin-4),A.T.C.C. Deposit Nos. 59394 and 59395 (which contain sequences encodingInterleukin-5), and A.T.C.C. Deposit No. 67153 (which contains sequencesencoding Interleukin-6).

Plasmids containing cytokine genes or immunomodulatory genes(International Publication Nos. WO 94/02951 and WO 96/21015, both ofwhich are incorporated by reference in their entirety) can be digestedwith appropriate restriction enzymes, and DNA fragments containing theparticular gene of interest can be inserted into a gene transfer vectorusing standard molecular biology techniques. (See, e.g., Sambrook etal., supra., or Ausubel et al. (eds) Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience).

Exemplary hormones, growth factors and other proteins which are usefulfor long term expression are described, for example, in EuropeanPublication No. 0437478B1, entitled “Cyclodextrin-Peptide Complexes.”Nucleic acid sequences encoding a variety of hormones can be used,including those encoding human growth hormone, insulin, calcitonin,prolactin, follicle stimulating hormone (FSH), luteinizing hormone (LH),human chorionic gonadotropin (HCG), and thyroid stimulating hormone(TSH). A variety of different forms of IGF-1 and IGF-2 growth factorpolypeptides are also well known the art and can be incorporated intogene transfer vectors for long term expression in vivo. See, e.g.,European Patent No. 0123228B1, published for grant Sep. 19, 1993,entitled “Hybrid DNA Synthesis of Mature Insulin-like Growth Factors.”As an additional example, the long term in vivo expression of differentforms of fibroblast growth factor can also be effected employing thecompositions and methods of invention. See, e.g., U.S. Pat. Nos.5,464,774, 5,155,214, and 4,994,559 for a description of differentfibroblast growth factors.

Polynucleotide sequences coding for the above-described molecules can beobtained using recombinant methods, such as by screening cDNA andgenomic libraries from cells expressing the gene, or by deriving thegene from a vector known to include the same. For example, plasmidswhich contain sequences that encode altered cellular products may beobtained from a depository such as the A.T.C.C., or from commercialsources. Plasmids containing the nucleotide sequences of interest can bedigested with appropriate restriction enzymes, and DNA fragmentscontaining the nucleotide sequences can be inserted into a gene transfervector using standard molecular biology techniques.

Alternatively, cDNA sequences for use with the present invention may beobtained from cells which express or contain the sequences, usingstandard techniques, such as phenol extraction and PCR of cDNA orgenomic DNA. See, e.g., Sambrook et al., supra, for a description oftechniques used to obtain and isolate DNA. Briefly, mRNA from a cellwhich expresses the gene of interest can be reverse transcribed withreverse transcriptase using oligo-dT or random primers. The singlestranded cDNA may then be amplified by PCR (see U.S. Pat. Nos.4,683,202, 4,683,195 and 4,800,159, see also PCR Technology: Principlesand Applications for DNA Amplification, Erlich (ed.), Stockton Press,1989)) using oligonucleotide primers complementary to sequences oneither side of desired sequences.

The nucleotide sequence of interest can also be produced synthetically,rather than cloned, using a DNA synthesizer (e.g., an Applied BiosystemsModel 392 DNA Synthesizer, available from ABI, Foster City, Calif.). Thenucleotide sequence can be designed with the appropriate codons for theexpression product desired. The complete sequence is assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge (1981) Nature 292:756;Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311.

The synthetic expression cassettes of the present invention can beemployed in the construction of packaging cell lines for use withretroviral vectors.

One type of retrovirus, the murine leukemia virus, or “MLV”, has beenwidely utilized for gene therapy applications (see generally Mann et al.(Cell 33:153, 1993), Cane and Mulligan (Proc, Nat'l. Acad. Sci. USA81:6349, 1984), and Miller et al., Human Gene 2lerapy 1:5-14, 1990.

Lentiviral vectors typically, comprise a 5′ lentiviral LTR, a tRNAbinding site, a packaging signal, a promoter operably linked to one ormore genes of interest, an origin of second strand DNA synthesis and a3′ lentiviral LTR, wherein the lentiviral vector contains a nucleartransport element. The nuclear transport element may be located eitherupstream (5′) or downstream (3′) of a coding sequence of interest.Within certain embodiments, the nuclear transport element is not RRE.Within one embodiment the packaging signal is an extended packagingsignal. Within other embodiments the promoter is a tissue specificpromoter, or, alternatively, a promoter such as CMV. Within otherembodiments, the lentiviral vector further comprises an internalribosome entry site.

A wide variety of lentiviruses may be utilized within the context of thepresent invention, including for example, lentiviruses selected from thegroup consisting of HIV, HIV-1, HIV-2, FIV and SIV.

In one embodiment of the present invention synthetic Env and/orGag-polymerase expression cassettes are provided comprising a promoterand a sequence encoding synthetic Gag-polymerase (SEQ ID NO:6) and atleast one of vpr, vpu, nef or vif, wherein the promoter is operablylinked to Gag-polymerase and vpr, vpu, nef or vif.

Within yet another aspect of the invention, host cells (e.g., packagingcell lines) are provided which contain any of the expression cassettesdescribed herein. For example, within one aspect packaging cell line areprovided comprising an expression cassette that comprises a sequenceencoding synthetic Env and/or Gag-polymerase, and a nuclear transportelement, wherein the promoter is operably linked to the sequenceencoding Env and/or Gag-polymerase. Packaging cell lines may furthercomprise a promoter and a sequence encoding tat, rev, or an envelope,wherein the promoter is operably linked to the sequence encoding tat,rev, or, the envelope. The packaging cell line may further comprise asequence encoding any one or more of nef, vif, vpu or vpr.

In one embodiment, the expression cassette (carrying, for example, thesynthetic Env, synthetic tat and/or synthetic Gag-polymerase) is stablyintegrated. The packaging cell line, upon introduction of a lentiviralvector, typically produces viral particles. The promoter regulatingexpression of the synthetic expression cassette may be inducible.Typically, the packaging cell line, upon introduction of a lentiviralvector, produces viral particles that are essentially free ofreplication competent virus.

Packaging cell lines are provided comprising an expression cassettewhich directs the expression of a synthetic Env (or Gag-polymerase)gene, an expression cassette which directs the expression of a Gag (orEnv) gene optimized for expression (e.g., Andre, S., et al., Journal ofVirology 72(2):1497-1503, 1998; Haas, J., et al., Current Biology6(3):315-324, 1996). A lentiviral vector is introduced into thepackaging cell line to produce a vector particle producing cell line.

As noted above, lentiviral vectors can be designed to carry or express aselected gene(s) or sequences of interest. Lentiviral vectors may bereadily constructed from a wide variety of lentiviruses (see RNA TumorViruses, Second Edition, Cold Spring Harbor Laboratory, 1985).Representative examples of lentiviruses included HIV, HIV-1, HIV-2, FIVand SIV. Such lentiviruses may either be obtained from patient isolates,or, more preferably, from depositories or collections such as theAmerican Type Culture Collection, or isolated from known sources usingavailable techniques.

Portions of the lentiviral gene delivery vectors (or vehicles) may bederived from different viruses. For example, in a given recombinantlentiviral vector, LTRs may be derived from an HIV, a packaging signalfrom SIV, and an origin of second strand synthesis from HrV-2.Lentiviral vector constructs may comprise a 5′ lentiviral LTR, a tRNAbinding site, a packaging signal, one or more heterologous sequences, anorigin of second strand DNA synthesis and a 3° LTR, wherein saidlentiviral vector contains a nuclear transport element that is not RRE.

Briefly, Long Terminal Repeats (“LTRs”) are subdivided into threeelements, designated U5, R and U3. These elements contain a variety ofsignals which are responsible for the biological activity of aretrovirus, including for example, promoter and enhancer elements whichare located within U3. LTRs may be readily identified in the provirus(integrated DNA form) due to their precise duplication at either end ofthe genome. As utilized herein, a 5′ LTR should be understood to includea 5′ promoter element and sufficient LTR sequence to allow reversetranscription and integration of the DNA form of the vector. The 3′ LTRshould be understood to include a polyadenylation signal, and sufficientLTR sequence to allow reverse transcription and integration of the DNAform of the vector.

The tRNA binding site and origin of second strand DNA synthesis are alsoimportant for a retrovirus to be biologically active, and may be readilyidentified by one of skill in the art. For example, retroviral tRNAbinds to a tRNA binding site by Watson-Crick base pairing, and iscarried with the retrovirus genome into a viral particle. The tRNA isthen utilized as a primer for DNA synthesis by reverse transcriptase.The tRNA binding site may be readily identified based upon its locationjust downstream from the 5′LTR. Similarly, the origin of second strandDNA synthesis is, as its name implies, important for the second strandDNA synthesis of a retrovirus. This region, which is also referred to asthe poly-purine tract, is located just upstream of the 3′LTR.

In addition to a 5′ and 3′ LTR, tRNA binding site, and origin of secondstrand DNA synthesis, recombinant retroviral vector constructs may alsocomprise a packaging signal, as well as one or more genes or codingsequences of interest. In addition, the lentiviral vectors have anuclear transport element which, in preferred embodiments is not RRE.Representative examples of suitable nuclear transport elements includethe element in Rous sarcoma virus (Ogert, et al., J. Virol. 70,3834-3843, 1996), the element in Rous sarcoma virus (Liu & Mertz, Genes& Dev., 9, 1766-1789, 1995) and the element in the genome of simianretrovirus type I (Zolotukhin, et al., J. Virol. 68, 7944-7952, 1994).Other potential elements include the elements in the histone gene(Kedes, Annu. Rev. Biochem. 48, 837-870, 1970), the α-interferon gene(Nagata et al., Nature 287, 401-408, 1980), the β-adrenergic receptorgene (Koilka, et al., Nature 329, 75-79, 1987), and the c-Jun gene(Hattorie, et al., Proc. Natl. Acad. Sci. USA 85, 9148-9152, 1988).

Recombinant lentiviral vector constructs typically lack bothGag-polymerase and env coding sequences. Recombinant lentiviral vectortypically contain less than 20, preferably 15, more preferably 10, andmost preferably 8 consecutive nucleotides found in Gag-polymerase or envgenes. One advantage of the present invention is that the syntheticGag-polymerase expression cassettes, which can be used to constructpackaging cell lines for the recombinant retroviral vector constructs,have little homology to wild-type Gag-polymerase sequences and thusconsiderably reduce or eliminate the possibility of homologousrecombination between the synthetic and wild-type sequences.

Lentiviral vectors may also include tissue-specific promoters to driveexpression of one or more genes or sequences of interest. For example,lentiviral vector particles of the invention can contain a liverspecific promoter to maximize the potential for liver specificexpression of the exogenous DNA sequence contained in the vectors.Preferred liver specific promoters include the hepatitis B X-genepromoter and the hepatitis B core protein promoter. These liver specificpromoters are preferably employed with their respective enhancers. Theenhancer element can be linked at either the 5′ or the 3′ end of thenucleic acid encoding the sequences of interest. The hepatitis B X genepromoter and its enhancer can be obtained from the viral genome as a 332base pair EcoRV-NcoI DNA fragment employing the methods described inTwu, et al., J. Virol. 61:3448-3453, 1987. The hepatitis B core proteinpromoter can be obtained from the viral genome as a 584 base pairBamHI-BglII DNA fragment employing the methods described in Gerlach, etal., Virol 189:59-66, 1992. It may be necessary to remove the negativeregulatory sequence in the BamHI-BglII fragment prior to inserting it.Other liver specific promoters include the AFP (alpha fetal protein)gene promoter and the albumin gene promoter, as disclosed in EP PatentPublication 0 415 731, the −1 antitrypsin gene promoter, as disclosed inRettenger, et al., Proc. Natl. Acad. Sci. 91:1460-1464, 1994, thefibrinogen gene promoter, the APO-A1 (Apolipoprotein A1) gene promoter,and the promoter genes for liver transference enzymes such as, forexample, SGOT, SGPT and glutamyle transferase. See also PCT PatentPublications WO 90/07936 and WO 91/02805 for a description of the use ofliver specific promoters in lentiviral vector particles.

Lentiviral vector constructs may be generated such that more than onegene of interest is expressed. This may be accomplished through the useof di- or oligo-cistronic cassettes (e.g., where the coding regions areseparated by 80 nucleotides or less, see generally Levin et al., Gene108:167-174, 1991), or through the use of Internal Ribosome Entry Sites(“IRES”).

Packaging cell lines suitable for use with the above describedrecombinant retroviral vector constructs may be readily prepared giventhe disclosure provided herein. Briefly, the parent cell line from whichthe packaging cell line is derived can be selected from a variety ofmammalian cell lines, including for example, 293, RD, COS-7, CHO, BHK,VERO, HT1080, and myeloma cells.

After selection of a suitable host cell for the generation of apackaging cell line, one or more expression cassettes are introducedinto the cell line in order to complement or supply in trans componentsof the vector which have been deleted.

Representative examples of suitable expression cassettes have beendescribed herein and include synthetic Env, tat, Gag, syntheticGag-protease, synthetic Gag-reverse transcriptase and syntheticGag-polymerase expression cassettes, which comprise a promoter and asequence encoding, e.g., Env, tat, or Gag-polymerase and at least one ofvpr, vpu, nef or vif, wherein the promoter is operably linked to Env,tat or Gag-polymerase and vpr, vpu, nef or vif. As described above,optimized Env, Gag and/or tat coding sequences may also be utilized invarious combinations in the generation of packaging cell lines.

Utilizing the above-described expression cassettes, a wide variety ofpackaging cell lines can be generated. For example, within one aspectpackaging cell line are provided comprising an expression cassette thatcomprises a sequence encoding synthetic HIV (e.g., Gag, Env, tat,Gag-polymerase, Gag-reverse transcriptase or Gag-protease) polypeptide,and a nuclear transport element, wherein the promoter is operably linkedto the sequence encoding the HIV polypeptide. Within other aspects,packaging cell lines are provided comprising a promoter and a sequenceencoding Gag, tat, rev, or an envelope (e.g., HIV env), wherein thepromoter is operably linked to the sequence encoding Gag, tat, rev, or,the envelope. Within further embodiments, the packaging cell line maycomprise a sequence encoding any one or more of nef, vif, vpu or vpr.For example, the packaging cell line may contain only nef, vif, vpu, orvpr alone, nef and vif, nef and vpu, nef and vpr, vif and vpu, vif andvpr, vpu and vpr, nef vif and vpu, nef vif and vpr, nef vpu and vpr,vvir vpu and vpr, or, all four of nef vif vpu and vpr.

In one embodiment, the expression cassette is stably integrated. Withinanother embodiment, the packaging cell line, upon introduction of alentiviral vector, produces particles. Within further embodiments thepromoter is inducible. Within certain preferred embodiments of theinvention, the packaging cell line, upon introduction of a lentiviralvector, produces particles that are free of replication competent virus.

The synthetic cassettes containing optimized coding sequences aretransfected into a selected cell line. Transfected cells are selectedthat (i) carry, typically, integrated, stable copies of the Gag, Pol,and Env coding sequences, and (ii) are expressing acceptable levels ofthese polypeptides (expression can be evaluated by methods known in theprior art, e.g., see Examples 1-4). The ability of the cell line toproduce VLPs may also be verified (Examples 6, 7 and 15).

A sequence of interest is constructed into a suitable viral vector asdiscussed above. This defective virus is then transfected into thepackaging cell line. The packaging cell line provides the viralfunctions necessary for producing virus-like particles into which thedefective viral genome, containing the sequence of interest, arepackaged. These VLPs are then isolated and can be used, for example, ingene delivery or gene therapy.

Further, such packaging cell lines can also be used to produce VLPsalone, which can, for example, be used as adjuvants for administrationwith other antigens or in vaccine compositions. Also, co-expression of aselected sequence of interest encoding a polypeptide (for example, anantigen) in the packaging cell line can also result in the entrapmentand/or association of the selected polypeptide in/with the VLPs.

2.3 DNA Immunization and Gene Delivery

A variety of polypeptide antigens can be used in the practice of thepresent invention. Polypeptide antigens can be included in DNAimmunization constructs containing, for example, any of the syntheticexpression cassettes described herein fused in-frame to a codingsequence for the polypeptide antigen, where expression of the constructresults in VLPs presenting the antigen of interest. Antigens can bederived from a wide variety of viruses, bacteria, fungi, plants,protozoans and other parasites. For example, the present invention willfind use for stimulating an immune response against a wide variety ofproteins from the herpesvirus family, including proteins derived fromherpes simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2 gB,gD, gH, VP16 and VP22; antigens derived from varicella zoster virus(VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) including CMVgB and gH; and antigens derived from other human herpesviruses such asHHV6 and HHV7. (See, e.g. Chee et al., Cytomegaloviruses (J. K.McDougall, ed., Springer-Verlag 1990) pp. 125-169, for a review of theprotein coding content of cytomegalovirus; McGeoch et al., J. Gen.Virol. (1988) 69:1531-1574, for a discussion of the various HSV-1encoded proteins; U.S. Pat. No. 5,171,568 for a discussion of HSV-1 andHSV-2 gB and gD proteins and the genes encoding therefore; Baer et al.,Nature (1984) 310:207-211, for the identification of protein codingsequences in an EBV genome; and Davison and Scott, J. Gen. Virol. (1986)67:1759-1816, for a review of VZV.)

Additionally, immune responses to antigens from the hepatitis family ofviruses, including hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis Evirus (HEV), and hepatitis G virus, can also be stimulated using theconstructs of the present invention. By way of example, the HCV genomeencodes several viral proteins, including E1 (also known as E) and E2(also known as E2/NSI), which will find use with the present invention(see, Houghton et al. Hepatology (1991) 14:381-388, for a discussion ofHCV proteins, including E1 and E2). The δ-antigen from HDV can also beused (see, e.g., U.S. Pat. No. 5,389,528, for a description of theδ-antigen).

Similarly, influenza virus is another example of a virus for which thepresent invention will be particularly useful. Specifically, theenvelope glycoproteins HA and NA of influenza A are of particularinterest for generating an immune response. Numerous HA subtypes ofinfluenza A have been identified (Kawaoka et al., Virology (1990)179:759-767; Webster et al. “Antigenic variation among type A influenzaviruses,” p. 127-168. In: P. Palese and D. W. Kingsbury (ed.), Geneticsof influenza viruses. Springer-Verlag, New York).

Other antigens of particular interest to be used in the practice of thepresent invention include antigens and polypeptides derived therefromfrom human papillomavirus (HPV), such as one or more of the variousearly proteins including E6 and E7; tick-borne encephalitis viruses; andHIV-1 (also known as HTLV-III, LAV, ARV, etc.), including, but notlimited to, antigens such as gp120, gp41, gp160, Gag and pol from avariety of isolates including, but not limited to, HIV_(IIIb),HIV_(SF2), HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV), HIV_(LAI), HIV_(MN),HIV-1_(CM235), HIV-1_(US4), other HIV-1 strains from diversesubtypes(e.g., subtypes, A through G, and O), HIV-2 strains and diversesubtypes (e.g., HIV-2_(UC1) and HIV-2_(UC2)). See, e.g., Myers, et al.,Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N.Mex.; Myers, et al., Human Retroviruses and Aids, 1990, Los Alamos, N.Mex.: Los Alamos National Laboratory.

Proteins derived from other viruses will also find use in the claimedmethods, such as without limitation, proteins from members of thefamilies Picornaviridae (e.g., polioviruses, etc.); Caliciviridae;Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae;Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabiesvirus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measlesvirus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g.,influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae;Retroviradae, e.g., HTLV-I; HTLV-II; HIV-1; HIV-2; simianimmunodeficiency virus (SIV) among others. See, e.g. Virology, 3rdEdition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B.N. Fields and D. M. Knipe, eds. 1991; Virology, 3rd Edition (Fields, BN, D M Knipe, P M Howley, Editors, 1996, Lippincott-Raven, Philadelphia,Pa.) for a description of these and other viruses.

Particularly preferred bacterial antigens are derived from organismsthat cause diphtheria, tetanus, pertussis, meningitis, and otherpathogenic states, including, without limitation, antigens derived fromCorynebacterium diphtheriae, Clostridium tetani, Bordetella pertusis,Neisseria meningitidis, including serotypes Meningococcus A, B, C, Y andWI35 (MenA, B, C, Y and WI35), Haemophilus influenza type B (Hib), andHelicobacter pylori. Examples of parasitic antigens include thosederived from organisms causing malaria, tuberculosis, and Lyme disease.

Furthermore, the methods described herein provide means for treating avariety of malignant cancers. For example, the system of the presentinvention can be used to enhance both humoral and cell-mediated immuneresponses to particular proteins specific to a cancer in question, suchas an activated oncogene, a fetal antigen, or an activation marker. Suchtumor antigens include any of the various MAGEs (melanoma associatedantigen E), including MAGE 1, 2, 3, 4, etc. (Boon, T. ScientificAmerican (March 1993):82-89); any of the various tyrosinases; MART 1(melanoma antigen recognized by T cells), mutant ras; mutant p53; p97melanoma antigen; CEA (carcinoembryonic antigen), among others.

DNA immunization using synthetic expression cassettes of the presentinvention has been demonstrated to be efficacious (Examples 8 and10-12). Animals were immunized with both the synthetic expressioncassette and the wild type expression cassette. The results of theimmunizations with plasmid-DNAs showed that the synthetic expressioncassettes provide a clear improvement of immunogenicity relative to thenative expression cassettes. Also, the second boost immunization induceda secondary immune response, for example after two to eight weeks.Further, the results of CTL assays showed increased potency of syntheticexpression cassettes for induction of cytotoxic T-lymphocyte (CTL)responses by DNA immunization.

It is readily apparent that the subject invention can be used to mountan immune response to a wide variety of antigens and hence to treat orprevent a large number of diseases.

2.3.1 Delivery of the Synthetic Expression Cassettes of the PresentInvention

Polynucleotide sequences coding for the above-described molecules can beobtained using recombinant methods, such as by screening cDNA andgenomic libraries from cells expressing the gene, or by deriving thegene from a vector known to include the same. The sequences can beanalyzed by conventional sequencing techniques. Furthermore, the desiredgene can be isolated directly from cells and tissues containing thesame, using standard techniques, such as phenol extraction and PCR ofcDNA or genomic DNA. See, e.g., Sambrook et al., supra, for adescription of techniques used to obtain, isolate and sequence DNA. Oncethe sequence is known, the gene of interest can also be producedsynthetically, rather than cloned. The nucleotide sequence can bedesigned with the appropriate codons for the particular amino acidsequence desired. In general, one will select preferred codons for theintended host in which the sequence will be expressed. The completesequence is assembled from overlapping oligonucleotides prepared bystandard methods and assembled into a complete coding sequence. See,e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984)223:1299; Jay et al., J. Biol. Chem. (1984) 259:6311; Stemmer, W. P.C.,(1995) Gene 164:49-53.

Next, the gene sequence encoding the desired antigen can be insertedinto a vector containing a synthetic expression cassette of the presentinvention (e.g., see Example 1 for construction of various exemplarysynthetic expression cassette). The antigen is inserted into thesynthetic coding sequence such that when the combined sequence isexpressed it results in the production of VLPs comprising thepolypeptide and/or the antigen of interest. Insertions can be madewithin the Gag coding sequence or at either end of the coding sequence(5′, amino terminus of the expressed polypeptide; or 3′, carboxyterminus of the expressed polypeptide—e.g., see Example 1)(Wagner, R.,et al., Arch Virol. 127:117-137, 1992; Wagner, R., et al., Virology200:162-175, 1994; Wu, X., et al., J. Virol. 69(6):3389-3398, 1995;Wang, C-T., et al., Virology 200:524-534, 1994; Chazal, N., et al.,Virology 68(1):111-122, 1994; Griffiths, J. C., et al., J. Virol.67(6):3191-3198, 1993; Reicin, A. S., et al., J. Virol. 69(2):642-650,1995).

Up to 50% of the coding sequences of p55Gag can be deleted withoutaffecting the assembly to virus-like particles and expression efficiency(Borsetti, A., et al, J. Virol. 72(11):9313-9317, 1998; Garnier, L., etal., J Virol 72(6):4667-4677, 1998; Zhang, Y., et al., J Virol72(3):1782-1789, 1998; Wang, C., et al., J Virol 72(10): 7950-7959,1998). In one embodiment of the present invention, immunogenicity of thehigh level expressing synthetic p55GagMod and p55GagProtMod expressioncassettes can be increased by the insertion of different structural ornon-structural HIV antigens, multiepitope cassettes, or cytokinesequences into deleted, mutated or truncated regions of p55GagModsequence. In another embodiment of the present invention, immunogenicityof the high level expressing synthetic Env expression cassettes can beincreased by the insertion of different structural or non-structural HIVantigens, multiepitope cassettes, or cytokine sequences into deletedregions of gp120Mod, gp140Mod or gp160Mod sequences. Such deletions maybe generated following the teachings of the present invention andinformation available to one of ordinary skill in the art. One possibleadvantage of this approach, relative to using full-length modified Envsequences fused to heterologous polypeptides, can be higherexpression/secretion efficiency and/or higher immunogenicity of theexpression product. Such deletions may be generated following theteachings of the present invention and information available to one ofordinary skill in the art. One possible advantage of this approach,relative to using full-length Env, Gag or Tat sequences fused toheterologous polypeptides, can be higher expression/secretion efficiencyand/or immunogenicity of the expression product.

When sequences are added to the amino terminal end of Gag (for example,when using the synthetic p55GagMod expression cassette of the presentinvention), the polynucleotide can contain coding sequences at the 5′end that encode a signal for addition of a myristic moiety to theGag-containing polypeptide (e.g., sequences that encode Met-Gly).

The ability of Gag-containing polypeptide constructs to form VLPs can beempirically determined following the teachings of the presentspecification.

HIV polypeptide/antigen synthetic expression cassettes include controlelements operably linked to the coding sequence, which allow for theexpression of the gene in vivo in the subject species. For example,typical promoters for mammalian cell expression include the SV40 earlypromoter, a CMV promoter such as the CMV immediate early promoter, themouse mammary tumor virus LTR promoter, the adenovirus major latepromoter (Ad MLP), and the herpes simplex virus promoter, among others.Other nonviral promoters, such as a promoter derived from the murinemetallothionein gene, will also find use for mammalian expression.Typically, transcription termination and polyadenylation sequences willalso be present, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al., supra, as well as a bovine growth hormoneterminator sequence.

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence.

Furthermore, plasmids can be constructed which include a chimericantigen-coding gene sequences, encoding, e.g., multipleantigens/epitopes of interest, for example derived from a single or frommore than one viral isolate.

Typically the antigen coding sequences precede or follow the syntheticcoding sequences and the chimeric transcription unit will have a singleopen reading frame encoding both the antigen of interest and thesynthetic Gag coding sequences. Alternatively, multi-cistronic cassettes(e.g., bi-cistronic cassettes) can be constructed allowing expression ofmultiple antigens from a single mRNA using the EMCV IRES, or the like.Lastly, antigens can be encoded on separate transcripts from independentpromoters on a single plasmid or other vector.

Once complete, the constructs are used for nucleic acid immunization orthe like using standard gene delivery protocols. Methods for genedelivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346,5,580,859, 5,589,466. Genes can be delivered either directly to thevertebrate subject or, alternatively, delivered ex vivo, to cellsderived from the subject and the cells reimplanted in the subject.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. Selected sequences can be insertedinto a vector and packaged in retroviral particles using techniquesknown in the art. The recombinant virus can then be isolated anddelivered to cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described (U.S. Pat. No. 5,219,740; Millerand Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human GeneTherapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burnset al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrieand Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

A number of adenovirus vectors have also been described. Unlikeretroviruses which integrate into the host genome, adenoviruses persistextrachromosomally thus minimizing the risks associated with insertionalmutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett etal., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human GeneTherapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barret al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988)6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).

Additionally, various adeno-associated virus (AAV) vector systems havebeen developed for gene delivery. AAV vectors can be readily constructedusing techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070(published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993);Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.,Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J.Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. CurrentTopics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. HumanGene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Another vector system useful for delivering the polynucleotides of thepresent invention is the enterically administered recombinant poxvirusvaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

Additional viral vectors which will find use for delivering the nucleicacid molecules encoding the antigens of interest include those derivedfrom the pox family of viruses, including vaccinia virus and avianpoxvirus. By way of example, vaccinia virus recombinants expressing thegenes can be constructed as follows. The DNA encoding the particularsynthetic Gag/antigen coding sequence is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the codingsequences of interest into the viral genome. The resulting TK⁻recombinant can be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the genes. Recombinant avipox viruses,expressing immunogens from mammalian pathogens, are known to conferprotective immunity when administered to non-avian species. The use ofan avipox vector is particularly desirable in human and other mammalianspecies since members of the avipox genus can only productivelyreplicate in susceptible avian species and therefore are not infectivein mammalian cells. Methods for producing recombinant avipoxviruses areknown in the art and employ genetic recombination, as described abovewith respect to the production of vaccinia viruses. See, e.g., WO91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery.

Members of the Alphavirus genus, such as, but not limited to, vectorsderived from the Sindbis, Semliki Forest, and Venezuelan EquineEncephalitis viruses, will also find use as viral vectors for deliveringthe polynucleotides of the present invention (for example, a syntheticGag- or Env-polypeptide encoding expression cassette as described inExample 14 below). For a description of Sindbis-virus derived vectorsuseful for the practice of the instant methods, see, Dubensky et al., J.Virol. (1996) 70:508-519; and International Publication Nos. WO 95/07995and WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No.5,843,723, issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S. Pat. No.5,789,245, issued Aug. 4, 1998, both herein incorporated by reference.

A vaccinia based infection/transfection system can be conveniently usedto provide for inducible, transient, expression of the coding sequencesof interest (for example, a synthetic Gag/HCV-core expression cassette)in a host cell. In this system, cells are first infected in vitro with avaccinia virus recombinant that encodes the bacteriophage T7 RNApolymerase. This polymerase displays exquisite specificity in that itonly transcribes templates bearing T7 promoters. Following infection,cells are transfected with the polynucleotide of interest, driven by aT7 promoter. The polymerase expressed in the cytoplasm from the vacciniavirus recombinant transcribes the transfected DNA into RNA which is thentranslated into protein by the host translational machinery. The methodprovides for high level, transient, cytoplasmic production of largequantities of RNA and its translation products. See, e.g., Elroy-Steinand Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al.,Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

As an alternative approach to infection with vaccinia or avipox virusrecombinants, or to the delivery of genes using other viral vectors, anamplification system can be used that will lead to high level expressionfollowing introduction into host cells. Specifically, a T7 RNApolymerase promoter preceding the coding region for T7 RNA polymerasecan be engineered. Translation of RNA derived from this template willgenerate T7 RNA polymerase which in turn will transcribe more template.Concomitantly, there will be a cDNA whose expression is under thecontrol of the T7 promoter. Thus, some of the T7 RNA polymerasegenerated from translation of the amplification template RNA will leadto transcription of the desired gene. Because some T7 RNA polymerase isrequired to initiate the amplification, T7 RNA polymerase can beintroduced into cells along with the template(s) to prime thetranscription reaction. The polymerase can be introduced as a protein oron a plasmid encoding the RNA polymerase. For a further discussion of T7systems and their use for transforming cells, see, e.g., InternationalPublication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986)189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al.,Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc.Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994)22:2114-2120; and U.S. Pat. No. 5,135,855.

The synthetic expression cassette of interest can also be deliveredwithout a viral vector. For example, the synthetic expression cassettecan be packaged as DNA or RNA in liposomes prior to delivery to thesubject or to cells derived therefrom. Lipid encapsulation is generallyaccomplished using liposomes which are able to stably bind or entrap andretain nucleic acid. The ratio of condensed DNA to lipid preparation canvary but will generally be around 1:1 (mg DNA:micromoles lipid), or moreof lipid. For a review of the use of liposomes as carriers for deliveryof nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991)1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol.101, pp. 512-527.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416);mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081);and purified transcription factors (Debs et al., J. Biol. Chem. (1990)265:10189-10192), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N. Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include (DDAB/DOPE)and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be preparedfrom readily available materials using techniques well known in the art.See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198;PCT Publication No. WO 90/11092 for a description of the synthesis ofDOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as,from Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY(1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA(1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta(1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA(1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szokaand Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; andSchaefer-Ridder et al., Science (1982) 215:166.

The DNA and/or protein antigen(s) can also be delivered in cochleatelipid compositions similar to those described by Papahadjopoulos et al.,Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos.4,663,161 and 4,871,488.

The synthetic expression cassette of interest (e.g., any of thesynthetic expression cassettes described in Example 1) may also beencapsulated, adsorbed to, or associated with, particulate carriers.Such carriers present multiple copies of a selected antigen to theimmune system and promote migration, trapping and retention of antigensin local lymph nodes. The particles can be taken up by professionantigen presenting cells such as macrophages and dendritic cells, and/orcan enhance antigen presentation through other mechanisms such asstimulation of cytokine release. Examples of particulate carriersinclude those derived from polymethyl methacrylate polymers, as well asmicroparticles derived from poly(lactides) andpoly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al.,Pharm. Res. (1993) 10:362-368; McGee J P, et al., J. Microencapsul.14(2):197-210, 1997; O'Hagan D T, et al., Vaccine 11(2):149-54, 1993.

Furthermore, other particulate systems and polymers can be used for thein vivo or ex vivo delivery of the gene of interest. For example,polymers such as polylysine, polyarginine, polyornithine, spermine,spermidine, as well as conjugates of these molecules, are useful fortransferring a nucleic acid of interest. Similarly, DEAEdextran-mediated transfection, calcium phosphate precipitation orprecipitation using other insoluble inorganic salts, such as strontiumphosphate, aluminum silicates including bentonite and kaolin, chromicoxide, magnesium silicate, talc, and the like, will find use with thepresent methods. See, e.g., Feigner, P. L., Advanced Drug DeliveryReviews (1990) 5:163-187, for a review of delivery systems useful forgene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No.5,831,005, issued Nov. 3, 1998, herein incorporated by reference) mayalso be used for delivery of a construct of the present invention.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are especially useful for deliveringsynthetic expression cassettes of the present invention. The particlesare coated with the synthetic expression cassette(s) to be delivered andaccelerated to high velocity, generally under a reduced atmosphere,using a gun powder discharge from a “gene gun.” For a description ofsuch techniques, and apparatuses useful therefore, see, e.g., U.S. Pat.Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and5,478,744. Also, needle-less injection systems can be used (Davis, H.L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

Recombinant vectors carrying a synthetic expression cassette of thepresent invention are formulated into compositions for delivery to thevertebrate subject. These compositions may either be prophylactic (toprevent infection) or therapeutic (to treat disease after infection).The compositions will comprise a “therapeutically effective amount” ofthe gene of interest such that an amount of the antigen can be producedin vivo so that an immune response is generated in the individual towhich it is administered. The exact amount necessary will vary dependingon the subject being treated; the age and general condition of thesubject to be treated; the capacity of the subject's immune system tosynthesize antibodies; the degree of protection desired; the severity ofthe condition being treated; the particular antigen selected and itsmode of administration, among other factors. An appropriate effectiveamount can be readily determined by one of skill in the art. Thus, a“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials.

The compositions will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, surfactants and the like, may be present in suchvehicles. Certain facilitators of immunogenicity or of nucleic aciduptake and/or expression can also be included in the compositions orcoadministered, such as, but not limited to, bupivacaine, cardiotoxinand sucrose.

Once formulated, the compositions of the invention can be administereddirectly to the subject (e.g., as described above) or, alternatively,delivered ex vivo, to cells derived from the subject, using methods suchas those described above. For example, methods for the ex vivo deliveryand reimplantation of transformed cells into a subject are known in theart and can include, e.g., dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, lipofectamineand LT-1 mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) (with or without thecorresponding antigen) in liposomes, and direct microinjection of theDNA into nuclei.

Direct delivery of synthetic expression cassette compositions in vivowill generally be accomplished with or without viral vectors, asdescribed above, by injection using either a conventional syringe,needless devices such as Bioject® or a gene gun, such as the Accell®gene delivery system (PowderJect Technologies, Inc., Oxford, England).The constructs can be delivered (e.g., injected) either subcutaneously,epidermally, intradermally, intramuscularly, intravenous, intramucosally(such as nasally, rectally and vaginally), intraperitoneally or orally.Delivery of DNA into cells of the epidermis is particularly preferred asthis mode of administration provides access to skin-associated lymphoidcells and provides for a transient presence of DNA in the recipient.Other modes of administration include oral ingestion and pulmonaryadministration, suppositories, needle-less injection, transcutaneous andtransdermal applications. Dosage treatment may be a single dose scheduleor a multiple dose schedule.

2.3.2 Ex Vivo Delivery of the Synthetic Expression Cassettes of thePresent Invention

In one embodiment, T cells, and related cell types (including but notlimited to antigen presenting cells, such as, macrophage, monocytes,lymphoid cells, dendritic cells, B-cells, T-cells, stem cells, andprogenitor cells thereof), can be used for ex vivo delivery of thesynthetic expression cassettes of the present invention. T cells can beisolated from peripheral blood lymphocytes (PBLs) by a variety ofprocedures known to those skilled in the art. For example, T cellpopulations can be “enriched” from a population of PBLs through theremoval of accessory and B cells. In particular, T cell enrichment canbe accomplished by the elimination of non-T cells using anti-MHC classII monoclonal antibodies. Similarly, other antibodies can be used todeplete specific populations of non-T cells. For example, anti-Igantibody molecules can be used to deplete B cells and anti-MacI antibodymolecules can be used to deplete macrophages.

T cells can be further fractionated into a number of differentsubpopulations by techniques known to those skilled in the art. Twomajor subpopulations can be isolated based on their differentialexpression of the cell surface markers CD4 and CD8. For example,following the enrichment of T cells as described above, CD4⁺ cells canbe enriched using antibodies specific for CD4 (see Coligan et al.,supra). The antibodies may be coupled to a solid support such asmagnetic beads. Conversely, CD8+ cells can be enriched through the useof antibodies specific for CD4 (to remove CD4⁺ cells), or can beisolated by the use of CD8 antibodies coupled to a solid support. CD4lymphocytes from HIV-1 infected patients can be expanded ex vivo, beforeor after transduction as described by Wilson et. al. (1995) J. Infect.Dis. 172:88.

Following purification of T cells, a variety of methods of geneticmodification known to those skilled in the art can be performed usingnon-viral or viral-based gene transfer vectors constructed as describedherein. For example, one such approach involves transduction of thepurified T cell population with vector-containing supernatant ofcultures derived from vector producing cells. A second approach involvesco-cultivation of an irradiated monolayer of vector-producing cells withthe purified T cells. A third approach involves a similar co-cultivationapproach; however, the purified T cells are pre-stimulated with variouscytokines and cultured 48 hours prior to the co-cultivation with theirradiated vector producing cells. Pre-stimulation prior to suchtransduction increases effective gene transfer (Nolta et al. (1992) Exp.Hematol. 20:1065). Stimulation of these cultures to proliferate alsoprovides increased cell populations for re-infusion into the patient.Subsequent to co-cultivation, T cells are collected from the vectorproducing cell monolayer, expanded, and frozen in liquid nitrogen.

Gene transfer vectors, containing one or more synthetic expressioncassette of the present invention (associated with appropriate controlelements for delivery to the isolated T cells) can be assembled usingknown methods.

Selectable markers can also be used in the construction of gene transfervectors. For example, a marker can be used which imparts to a mammaliancell transduced with the gene transfer vector resistance to a cytotoxicagent. The cytotoxic agent can be, but is not limited to, neomycin,aminoglycoside, tetracycline, chloramphenicol, sulfonamide, actinomycin,netropsin, distamycin A, anthracycline, or pyrazinamide. For example,neomycin phosphotransferase II imparts resistance to the neomycinanalogue geneticin (G418).

The T cells can also be maintained in a medium containing at least onetype of growth factor prior to being selected. A variety of growthfactors are known in the art which sustain the growth of a particularcell type. Examples of such growth factors are cytokine mitogens such asrIL-2, IL-10, IL-12, and IL-15, which promote growth and activation oflymphocytes. Certain types of cells are stimulated by other growthfactors such as hormones, including human chorionic gonadotropin (hCG)and human growth hormone. The selection of an appropriate growth factorfor a particular cell population is readily accomplished by one of skillin the art.

For example, white blood cells such as differentiated progenitor andstem cells are stimulated by a variety of growth factors. Moreparticularly, IL-3, IL-4, IL-5, IL-6, IL-9, GM-CSF, M-CSF, and G-CSF,produced by activated T_(H) and activated macrophages, stimulate myeloidstem cells, which then differentiate into pluripotent stem cells,granulocyte-monocyte progenitors, eosinophil progenitors, basophilprogenitors, megakaryocytes, and erythroid progenitors. Differentiationis modulated by growth factors such as GM-CSF, IL-3, IL-6, IL-11, andEPO.

Pluripotent stem cells then differentiate into lymphoid stem cells, bonemarrow stromal cells, T cell progenitors, B cell progenitors,thymocytes, T_(H) Cells, T_(C) cells, and B cells. This differentiationis modulated by growth factors such as IL-3, IL-4, IL-6, IL-7, GM-CSF,M-CSF, G-CSF, IL-2, and IL-5.

Granulocyte-monocyte progenitors differentiate to monocytes,macrophages, and neutrophils. Such differentiation is modulated by thegrowth factors GM-CSF, M-CSF, and IL-8. Eosinophil progenitorsdifferentiate into eosinophils. This process is modulated by GM-CSF andIL-5.

The differentiation of basophil progenitors into mast cells andbasophils is modulated by GM-CSF, IL-4, and IL-9. Megakaryocytes produceplatelets in response to GM-CSF, EPO, and IL-6. Erythroid progenitorcells differentiate into red blood cells in response to EPO.

Thus, during activation by the CD3-binding agent, T cells can also becontacted with a mitogen, for example a cytokine such as IL-2. Inparticularly preferred embodiments, the IL-2 is added to the populationof T cells at a concentration of about 50 to 100 μg/ml. Activation withthe CD3-binding agent can be carried out for 2 to 4 days.

Once suitably activated, the T cells are genetically modified bycontacting the same with a suitable gene transfer vector underconditions that allow for transfection of the vectors into the T cells.Genetic modification is carried out when the cell density of the T cellpopulation is between about 0.1×10⁶ and 5×10⁶, preferably between about0.5×10⁶ and 2×10⁶. A number of suitable viral and nonviral-based genetransfer vectors have been described for use herein.

After transduction, transduced cells are selected away fromnon-transduced cells using known techniques. For example, if the genetransfer vector used in the transduction includes a selectable markerwhich confers resistance to a cytotoxic agent, the cells can becontacted with the appropriate cytotoxic agent, whereby non-transducedcells can be negatively selected away from the transduced cells. If theselectable marker is a cell surface marker, the cells can be contactedwith a binding agent specific for the particular cell surface marker,whereby the transduced cells can be positively selected away from thepopulation. The selection step can also entail fluorescence-activatedcell sorting (FACS) techniques, such as where FACS is used to selectcells from the population containing a particular surface marker, or theselection step can entail the use of magnetically responsive particlesas retrievable supports for target cell capture and/or backgroundremoval.

More particularly, positive selection of the transduced cells can beperformed using a FACS cell sorter (e.g. a FACSVantage™ Cell Sorter,Becton Dickinson Immunocytometry Systems, San Jose, Calif.) to sort andcollect transduced cells expressing a selectable cell surface marker.Following transduction, the cells are stained with fluorescent-labeledantibody molecules directed against the particular cell surface marker.The amount of bound antibody on each cell can be measured by passingdroplets containing the cells through the cell sorter. By imparting anelectromagnetic charge to droplets containing the stained cells, thetransduced cells can be separated from other cells. The positivelyselected cells are then harvested in sterile collection vessels. Thesecell sorting procedures are described in detail, for example, in theFACSVantage™ Training Manual, with particular reference to sections 3-11to 3-28 and 10-1 to 10-17.

Positive selection of the transduced cells can also be performed usingmagnetic separation of cells based on expression or a particular cellsurface marker. In such separation techniques, cells to be positivelyselected are first contacted with specific binding agent (e.g., anantibody or reagent the interacts specifically with the cell surfacemarker). The cells are then contacted with retrievable particles (e.g.,magnetically responsive particles) which are coupled with a reagent thatbinds the specific binding agent (that has bound to the positive cells).The cell-binding agent-particle complex can then be physically separatedfrom non-labeled cells, for example using a magnetic field. When usingmagnetically responsive particles, the labeled cells can be retained ina container using a magnetic filed while the negative cells are removed.These and similar separation procedures are known to those of ordinaryskill in the art.

Expression of the vector in the selected transduced cells can beassessed by a number of assays known to those skilled in the art. Forexample, Western blot or Northern analysis can be employed depending onthe nature of the inserted nucleotide sequence of interest. Onceexpression has been established and the transformed T cells have beentested for the presence of the selected synthetic expression cassette,they are ready for infusion into a patient via the peripheral bloodstream.

The invention includes a kit for genetic modification of an ex vivopopulation of primary mammalian cells. The kit typically contains a genetransfer vector coding for at least one selectable marker and at leastone synthetic expression cassette contained in one or more containers,ancillary reagents or hardware, and instructions for use of the kit.

EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Generation of Synthetic Gag and Env Expression Cassettes A.Modification of HIV-1 Gag, Gag-Protease, Gag-Reverse Transcriptase andGag-Polymerase Nucleic Acid Coding Sequences

The Gag (SEQ ID NO:1), Gag-protease (SEQ ID NO:2), Gag-polymerase (SEQID NO:3), and Gag-reverse transcriptase (SEQ ID NO:77) coding sequenceswere selected from the HIV-1SF2 strain (Sanchez-Pescador, R., et al.,Science 227(4686): 484-492, 1985; Luciw, P. A., et al. U.S. Pat. No.5,156,949, issued Oct. 20, 1992, herein incorporated by reference;Luciw, P. A., et al., U.S. Pat. No. 5,688,688, Nov. 18, 1997). Thesesequences were manipulated to maximize expression of their geneproducts.

First, the HIV-1 codon usage pattern was modified so that the resultingnucleic acid coding sequence was comparable to codon usage found inhighly expressed human genes. The HIV codon usage reflects a highcontent of the nucleotides A or T of the codon-triplet. The effect ofthe HIV-1 codon usage is a high AT content in the DNA sequence thatresults in a high AU content in the RNA and in a decreased translationability and instability of the mRNA. In comparison, highly expressedhuman codons prefer the nucleotides G or C. The Gag-encoding sequenceswere modified to be comparable to codon usage found in highly expressedhuman genes.

FIG. 11 presents a comparison of the percent A-T content for the cDNAsof stable versus unstable RNAs (comparison window size=50). Human IFNγmRNA is known to (i) be unstable, (ii) have a short half-life, and (iii)have a high A-U content. Human GAPDH (glyceraldehyde-3-phosphatedehydrogenase) mRNA is known to (i) be a stable RNA, and (i) have a lowA-U content. In FIG. 11, the percent A-T content of these two sequencesare compared to the percent A-T content of native HIV-1SF2 Gag cDNA andto the synthetic Gag cDNA sequence of the present invention. The top twopanels of the figure show the percent A-T content over the length of thesequences for IFNγ and native Gag. The bottom two panels of the figureshow the percent A-T content over the length of the sequences for GAPDHand the synthetic Gag. Experiments performed in support of the presentinvention showed that the synthetic Gag sequences were capable of higherlevel of protein production (see the Examples) than the native Gagsequences. The data in FIG. 11 suggest that one reason for thisincreased production may be increased stability of the mRNAcorresponding to the synthetic Gag coding sequences versus the mRNAcorresponding to the native Gag coding sequences.

Second, there are inhibitory (or instability) elements (INS) locatedwithin the coding sequences of the Gag and Gag-protease coding sequences(Schneider R, et al., J. Virol. 71(7):4892-4903, 1997). RRE is asecondary RNA structure that interacts with the HIV encoded Rev-proteinto overcome the expression down-regulating effects of the INS. Toovercome the requirement for post-transcriptional activating mechanismsof RRE and Rev, and to enhance independent expression of the Gagpolypeptide, the INS were inactivated by introducing multiple pointmutations that did not alter the reading frame of the encoded proteins.FIG. 1 shows the original SF2 Gag sequence, the location of the INSsequences, and the modifications made to the INS sequences to reducetheir effects.

For the Gag-protease sequence (wild type, SEQ ID NO:2; synthetic, SEQ IDNOs:5, 78 and 79), the changes in codon usage were restricted to theregions up to the −1 frameshift and starting again at the end of the Gagreading frame (FIG. 2; the region indicated in lower case letters inFIG. 2 is the unmodified region). Further, inhibitory (or instability)elements (INS) located within the coding sequences of the Gag-proteasepolypeptide coding sequence were altered as well (indicated in FIG. 2).The synthetic coding sequences were assembled by the Midland CertifiedReagent Company (Midland, Tex.).

Modification of the Gag-polymerase sequences (wild type, SEQ ID NO:3;synthetic, SEQ ID NO:6) and Gag-reverse transcriptase sequences (SEQ IDNOs:80 through 84) include similar modifications as described forGag-protease in order to preserve the frameshift region. Locations ofthe inactivation sites and changes to the sequence to alter theinactivation sites are presented in FIG. 12 for the native HIV-1_(SF2)Gag-polymerase sequence.

In one embodiment of the invention, the full length polymerase codingregion of the Gag-polymerase sequence is included with the synthetic Gagsequences in order to increase the number of epitopes for virus-likeparticles expressed by the synthetic, optimized Gag expression cassette.Because synthetic HIV-1 Gag-polymerase expresses the potentiallydeleterious functional enzymes reverse transcriptase (RT) and integrase(INT) (in addition to the structural proteins and protease), it isimportant to inactivate RT and INT functions. Several in-frame deletionsin the RT and INT reading frame can be made to achieve catalyticnonfunctional enzymes with respect to their RT and INT activity. {Jay.A. Levy (Editor) (1995) The Retroviridae, Plenum Press, New York. ISBN0-306-45033X. Pages 215-20; Grimison, B. and Laurence, J. (1995),Journal Of Acquired Immune Deficiency Syndromes and Human Retrovirology9(1):58-68; Wakefield, J. K., et al., (1992) Journal Of Virology66(11):6806-6812; Esnouf, R., et al., (1995) Nature Structural Biology2(4):303-308; Maignan, S., et al., (1998) Journal Of Molecular Biology282(2):359-368; Katz, R. A. and Skalka, A. M. (1994) Annual Review OfBiochemistry 73 (1994); Jacobo-Molina, A., et al., (1993) Proceedings Ofthe National Academy Of Sciences Of the United States Of America90(13):6320-6324; Hickman, A. B., et al., (1994) Journal Of BiologicalChemistry 269(46):29279-29287; Goldgur, Y., et al., (1998) ProceedingsOf the National Academy Of Sciences Of the United States Of America95(16):9150-9154; Goette, M., et al., (1998) Journal Of BiologicalChemistry 273(17):10139-10146; Gorton, J. L., et al., (1998) Journal ofVirology 72(6):5046-5055; Engelman, A., et al., (1997) Journal OfVirology 71(5):3507-3514; Dyda, F., et al., Science 266(5193):1981-1986;Davies, J. F., et al., (1991) Science 252(5002):88-95; Bujacz, G., etal., (1996) Febs Letters 398(2-3):175-178; Beard, W. A., et al., (1996)Journal Of Biological Chemistry 271(21):12213-12220; Kohlstaedt, L. A.,et al., (1992) Science 256(5065):1783-1790; Krug, M. S, and Berger, S.L. (1991) Biochemistry 30(44):10614-10623; Mazumder, A., et al., (1996)Molecular Pharmacology 49(4):621-628; Palaniappan, C., et al., (1997)Journal Of Biological Chemistry 272(17):11157-11164; Rodgers, D. W., etal., (1995) Proceedings Of the National Academy Of Sciences Of theUnited States Of America 92(4):1222-1226; Sheng, N. and Dennis, D.(1993) Biochemistry 32(18):4938-4942; Spence, R. A., et al., (1995)Science 267(5200):988-993.}

Furthermore selected B- and/or T-cell epitopes can be added to theGag-polymerase constructs within the deletions of the RT- and INT-codingsequence to replace and augment any epitopes deleted by the functionalmodifications of RT and INT. Alternately, selected B- and T-cellepitopes (including CTL epitopes) from RT and INT can be included in aminimal VLP formed by expression of the synthetic Gag or syntheticGagProt cassette, described above. (For descriptions of known HIV B- andT-cell epitopes see, HIV Molecular Immunology Database CTL SearchInterface; Los Alamos Sequence Compendia, 1987-1997;Internet address:http://hiv-web.lan1.gov/immunology/index.html.)

The resulting modified coding sequences are presented as a synthetic Gagexpression cassette (SEQ ID NO:4), a synthetic Gag-protease expressioncassette (SEQ ID NOs:5, 78 and 79), and a synthetic Gag-polymeraseexpression cassette (SEQ ID NO:6). Synthetic expression cassettescontaining codon modifications in the reverse transcriptase region areshown in SEQ ID NOs:80 through 84. An alignment of selected sequences ispresented in FIG. 7. A common region (Gag-common; SEQ ID NO:9) extendsfrom position 1 to position 1262.

The synthetic DNA fragments for Gag and Gag-protease were cloned intothe following expression vectors: pCMVKm2, for transient expressionassays and DNA immunization studies, the pCMVKm2 vector was derived frompCMV6a (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) andcomprises a kanamycin selectable marker, a ColE1 origin of replication,a CMV promoter enhancer and Intron A, followed by an insertion site forthe synthetic sequences described below followed by a polyadenylationsignal derived from bovine growth hormone—the pCMVKm2 vector differsfrom the pCMV-link vector only in that a polylinker site was insertedinto pCMVKm2 to generate pCMV-link (FIG. 14, polylinker at positions1646 to 1697); pESN2dhfr (FIG. 13A) and pCMVPLEdhfr (also known aspCMVIII as shown in FIG. 13B), for expression in Chinese Hamster Ovary(CHO) cells; and, pAcC13, a shuttle vector for use in the Baculovirusexpression system (pAcC13, was derived from pAcC12 which was describedby Munemitsu S., et al., Mol Cell Biol. 10(11):5977-5982, 1990).

A restriction map for vector pCMV-link is presented in FIG. 14. In thefigure, the CMV promoter (CMV IE ENH/PRO), bovine growth hormoneterminator (BGH pA), kanamycin selectable marker (kan), and a ColE1origin of replication (ColE1 ori) are indicated. A polycloning site isalso indicated in the figure following the CMV promoter sequences.

A restriction map for vector pESN2dhfr is presented in FIG. 13A. In thefigure, the CMV promoter (pCMV, hCMVIE), bovine growth hormoneterminator (BGHpA), SV40 origin of replication (SV40ori), neomycinselectable marker (Neo), SV40 polyA (SV40pA), Adenovirus 2 late promoter(Ad2VLP), and the murine dhfr gene (mu dhfr) are indicated. Apolycloning site is also indicated in the figure following the CMVpromoter sequences.

Briefly, construction of pCMVPLEdhfr (pCMVIII) was as follows. Toconstruct a DHFR cassette, the EMCV IRES (internal ribosome entry site)leader was PCR-amplified from pCite-4-a+ (Novagen, Inc., Milwaukee,Wis.) and inserted into pET-23d (Novagen, Inc., Milwaukee, Wis.) as anXba-Nco fragment to give pET-EMCV. The dhfr gene was PCR-amplified frompESN2dhfr to give a product with a Gly-Gly-Gly-Ser spacer in place ofthe translation stop codon and inserted as an Nco-BamH1 fragment to givepET-E-DHFR. Next, the attenuated neo gene was PCR amplified from apSV2Neo (Clontech, Palo Alto, Calif.) derivative and inserted into theunique BamH1 site of pET-E-DHFR to give pET-E-DHFR/Neo_((m2)). Then, thebovine growth hormone terminator from pcDNA3 (Invitrogen, Inc.,Carlsbad, Calif.) was inserted downstream of the neo gene to givepET-E-DHFR/Neo_((m2))BGHt. The EMCV-dhfr/neo selectable marker cassettefragment was prepared by cleavage of pET-E-DHFR/Neo_((m2))BGHt. The CMVenhancer/promoter plus Intron A was transferred from pCMV6a (Chapman etal., Nuc. Acids Res. (1991) 19:3979-3986) as a HindIII-SalI fragmentinto pUC19 (New England Biolabs, Inc., Beverly, Mass.). The vectorbackbone of pUC19 was deleted from the Nde1 to the Sap1 sites. The abovedescribed DHFR cassette was added to the construct such that the EMCVIRES followed the CMV promoter to produce the final construct. Thevector also contained an amp^(r) gene and an SV40 origin of replication.

Selected pCMVKm2 vectors containing the synthetic expression cassetteshave been designated as follows: pCMVKm2.GagMod.SF2,pCMVKm2.GagprotMod.SF2, and pCMVKm2.GagpolMod.SF2,pCMVKm2.GagprotMod.SF2.GP1 (SEQ ID NO:78) and pCMVKm2.GagprotMod.SF2.GP2(SEQ ID NO:79). Other exemplary Gag-encoding expressing cassettes areshown in the Figures and as Sequence Listings.

B. Modification of HIV-1 Gag/Hepatitis C Core Chimeric Protein NucleicAcid Coding Sequences Generation of Synthetic Expression Cassettes

To facilitate the ligation of the Gag and HCV core coding sequences, PCRamplification was employed. The synthetic p55Gag expression cassette wasused as a PCR template with the following primers: GAG5(SEQ ID NO:11)and P55-SAL3 (SEQ ID NO:12). The PCR amplification was conducted at 55°C. for 25 cycles using Stratagene's Pfu polymerase. The resulting PCRproduct was rendered free of nucleotides and primers using the PromegaPCR clean-up kit and then subjected to EcoRI and SalI digestions. ForHCV core coding sequences, the following primers were used with an HCVtemplate (Houghton, M., et al., U.S. Pat. No. 5,714,596, issued Feb. 3,1998; Houghton, M., et al., U.S. Pat. No. 5,712,088, issued Jan. 27,1998; Houghton, M., et al., U.S. Pat. No. 5,683,864, issued Nov. 4,1997; Weiner, A. J., et al., U.S. Pat. No. 5,728,520, issued Mar. 17,1998; Weiner, A. J., et al., U.S. Pat. No. 5,766,845, issued Jun. 16,1998; Weiner, A. J., et al., U.S. Pat. No. 5,670,152, issued Sep. 23,1997; all herein incorporated by reference): CORESAL 5 (SEQ ID NO:13)and 173CORE(SEQ ID NO:14) using the conditions outlined above. Thepurified product was digested with SalI and BamHI restriction enzymes.The digested Gag and HCV core PCR products were ligated into the pCMVKm2vector digested with EcoRI and BamHI. Ligation of the PCR products atthe SalI site resulted in a direct fusion of the final amino acid ofp55Gag to the second amino acid of HCV core, serine. Amino acid 173 ofcore is a serine and is followed immediately by a TAG termination codon.The sequence of the fusion clone was confirmed. The pCMVKm2 vectorcontaining the synthetic expression cassette was designated aspCMVKm2.GagModHCVcore.

The EcoRI-BamHI fragment of p55Gag-core 173 was also cloned intoEcoRI-BamHI-digested pAcC13 for baculovirus expression. Western blotsconfirmed expression and sucrose gradient sedimentation along withelectron microscopy confirmed particle formation. To generate the aboveclone but containing the synthetic Gag sequences (instead of wild-type),the following steps were performed: pCMVKm2-modified p55Gag was used astemplate for PCR amplification with MS65 (SEQ ID NO:15) and MS66(SEQ IDNO:16) primers. The region amplified corresponds to the BspHI and SalIsites at the C-terminus of synthetic Gag sequence. The amplificationproduct was digested with BspHI and SalI and ligated to SalI/BamHIdigested pCMV-link along with the Sal/BspHI fragment frompCMV-Km-p55modGag, representing the amino terminal end of modified Gag,and the SalI/BamHI fragment from pCMV-p55Gag-core173. Thereafter, aT4-blunted-SalI partial/BamHI fragment was ligated into pAcC4-SmaI/BamHIto generate pAcC4-p55GagMod-core173 (containing the synthetic sequencepresented as SEQ ID NO:7).

C. Defining of the Major Homology Region (MHR) of HIV-1 p55Gag

The Major Homology Region (MHR) of HIV-1 p55 (Gag) is located in thep24-CA sequence of Gag. It is a conserved stretch of 20 amino acids (SEQID NO:19). The position in the wild type HIV-1_(SF2) Gag protein is fromaa 286-305 and spans a region from nucleotides 856-915 in the nativeHIV-1_(SF2) Gag DNA-sequence. The position in the synthetic Gag proteinis from aa 288-307 and spans a region from nucleotides 862-921 for thesynthetic Gag DNA-sequence. The nucleotide sequence for the MHR in thesynthetic GagMod.SF2 is presented as SEQ ID NO:20. Mutations ordeletions in the amino acid sequence of the MHR can severely impairparticle production (Borsetti, A., et al., J. Virol. 72(11):9313-9317,1998; Mammano, F., et al., J Virol 68(8):4927-4936, 1994).

Percent identity to the MHR nucleotide sequence can be determined, forexample, using the MacDNAsis program (Hitachi Software EngineeringAmerica Limited, South San Francisco, Calif.), Higgins algorithm, withthe following exemplary parameters: gap penalty=5, no. of topdiagonals=5, fixed gap penalty=5, K-tuple=2, window size=5, and floatinggap penalty=10.

D. Generation of Synthetic Env Expression Cassettes

Env coding sequences of the present invention include, but are notlimited to, polynucleotide sequences encoding the following HIV-encodedpolypeptides: gp160, gp140, and gp120 (see, e.g., U.S. Pat. No.5,792,459 for a description of the HIV-1_(SF2) (“SF2”) Env polypeptide).The relationships between these polypeptides is shown schematically inFIG. 15 (in the figure: the polypeptides are indicated as lines, theamino and carboxy termini are indicated on the gp160 line; the opencircle represents the oligomerization domain; the open square representsa transmembrane spanning domain (TM); and “c” represents the location ofa cleavage site, in gp140.mut the “X” indicates that the cleavage sitehas been mutated such that it no longer functions as a cleavage site).The polypeptide gp160 includes the coding sequences for gp120 and gp41.The polypeptide gp41 is comprised of several domains including anoligomerization domain (OD) and a transmembrane spanning domain (TM). Inthe native envelope, the oligomerization domain is required for thenon-covalent association of three gp41 polypeptides to form a trimericstructure: through non-covalent interactions with the gp41 trimer (anditself), the gp120 polypeptides are also organized in a trimericstructure. A cleavage site (or cleavage sites) exists approximatelybetween the polypeptide sequences for gp120 and the polypeptidesequences corresponding to gp41. This cleavage site(s) can be mutated toprevent cleavage at the site. The resulting gp140 polypeptidecorresponds to a truncated form of gp160 where the transmembranespanning domain of gp41 has been deleted. This gp140 polypeptide canexist in both monomeric and oligomeric (i.e. trimeric) forms by virtueof the presence of the oligomerization domain in the gp41 moiety. In thesituation where the cleavage site has been mutated to prevent cleavageand the transmembrane portion of gp41 has been deleted the resultingpolypeptide product is designated “mutated” gp140 (e.g., gp140.mut). Aswill be apparent to those in the field, the cleavage site can be mutatedin a variety of ways. The native amino acid sequence in the SF162cleavage sites is: APTKAKRRVVQREKR (SEQ ID NO:21), where KAKRR (SEQ IDNO:22) is termed the “second” site and REKR (SEQ ID NO:23) is the “firstsite”. Exemplary mutations include the following constructs:gp140.mut7.modSF162 which encodes the amino acid sequenceAPTKAISSVVQSEKS (SEQ ID NO:24) in the cleavage site region;gp140.mut8.modSF162 which encodes the amino acid sequenceAPTIAISSVVQSEKS (SEQ ID NO:25) in the cleavage site region andgp140mut.modSF162 which encodes the amino acid sequence APTKAKRRVVQREKS(SEQ ID NO:26). Mutations are denoted in bold. The native amino acidsequence in the US4 cleavage sites is: APTQAKRRVVQREKR (SEQ ID NO:27),where QAKRR (SEQ ID NO:28) is termed the “second” site and REKR (SEQ IDNO:23) is the “first site”. Exemplary mutations include the followingconstruct: gp140.mut.modUS4 which encodes the amino acid sequenceAPTQAKRRVVQREKS (SEQ ID NO:29) in the cleavage site region. Mutationsare denoted in bold.

E. Modification of HIV-1 Env (Envelope) Nucleic Acid Coding Sequences

In one embodiment of the present invention, wild-type Env codingsequences were selected from the HIV-1_(SF162) (“SF162”) strain(Cheng-Mayer (1989) PNAS USA 86:8575-8579). These SF162 sequences wereas follows: gp120, SEQ ID NO:30 (FIG. 16); gp140, SEQ ID NO:31 (FIG.17); and gp160, SEQ ID NO:32 (FIG. 18).

In another embodiment of the present invention, wild-type Env codingsequences were selected from the HIV-US4 strain (Mascola, et al. (1994)J. Infect. Dis. 169:48-54). These US4 sequences were as follows: gp120,SEQ ID NO:51 (FIG. 38); gp140, SEQ ID NO:52 (FIG. 39); and gp160, SEQ IDNO:53 (FIG. 40).

These Env coding sequences were manipulated to maximize expression oftheir gene products.

First, the wild-type coding region was modified in one or more of thefollowing ways. In one embodiment, sequences encoding hypervariableregions of Env, particularly V1 and/or V2 were deleted. In otherembodiments, mutations were introduced into sequences encoding thecleavage site in Env to abrogate the enzymatic cleavage of oligomericgp140 into gp120 monomers. (See, e.g., Earl et al. (1990) PNAS USA87:648-652; Earl et al. (1991) J. Virol. 65:31-41). In yet otherembodiments, hypervariable region(s) were deleted, N-glycosylation siteswere removed and/or cleavage sites mutated.

Second, the HIV-1 codon usage pattern was modified so that the resultingnucleic acid coding sequence was comparable to codon usage found inhighly expressed human genes. The HIV codon usage reflects a highcontent of the nucleotides A or T in the codon-triplet. The effect ofthe HIV-1 codon usage is a high AT content in the DNA sequence thatresults in a decreased translation ability and instability of the mRNA.In comparison, highly expressed human codons prefer the nucleotides G orC. The Env coding sequences were modified to be comparable to codonusage found in highly expressed human genes.

FIGS. 22A-22H present comparisons of the percent A-T content for thecDNAs of stable versus unstable RNAs (comparison window size=50). HumanIFNγ mRNA is known to (i) be unstable, (ii) have a short half-life, and(iii) have a high A-U content. Human GAPDH (glyceraldehyde-3-phosphatedehydrogenase) mRNA is known to (i) be a stable RNA, and (i) have a lowA-U content. In FIGS. 22A-H, the percent A-T content of these twosequences are compared to the percent A-T content of (1) native HIV-1US4 Env gp160 cDNA, a synthetic US4 Env gp160 cDNA sequence (i.e.,having modified codons) of the present invention; and (2) native HIV-1SF162 Env gp160 cDNA, a synthetic SF162 Env gp160 cDNA sequence (i.e.,having modified codons) of the present invention. FIGS. 22A-H show thepercent A-T content over the length of the sequences for IFNγ (FIGS. 22Cand 22G); native gp160 Env US4 and SF162 (FIGS. 22A and 22E,respectively); GAPDH (FIGS. 22D and 22H); and the synthetic gp160 Envfor US4 and SF162 (FIGS. 22B and 22F). Experiments performed in supportof the present invention showed that the synthetic Env sequences werecapable of higher level of protein production (see the Examples) thanthe native Env sequences. The data in FIGS. 22A-H suggest that onereason for this increased production is increased stability of the mRNAcorresponding to the synthetic Env coding sequences versus the mRNAcorresponding to the native Env coding sequences.

To create the synthetic coding sequences of the present invention thegene cassettes were designed to comprise the entire coding sequence ofinterest. Synthetic gene cassettes were constructed by oligonucleotidesynthesis and PCR amplification to generate gene fragments. Primers werechosen to provide convenient restriction sites for subcloning. Theresulting fragments were then ligated to create the entire desiredsequence which was then cloned into an appropriate vector. The finalsynthetic sequences were (i) screened by restriction endonucleasedigestion and analysis, (ii) subjected to DNA sequencing in order toconfirm that the desired sequence had been obtained and (iii) theidentity and integrity of the expressed protein confirmed by SDS-PAGEand Western blotting (See, Examples. The synthetic coding sequences wereassembled at Chiron Corp. or by the Midland Certified Reagent Company(Midland, Tex.).

Exemplary modified coding sequences are presented as synthetic Envexpression cassettes in Table 1A and 1B. The following expressioncassettes (i) have unique, terminal EcoRI and XbaI cloning sites; (ii)include Kozak sequences to promote optimal translation; (iii) tPA signalsequences (to direct the ENV polypeptide to the cell membrane, see,e.g., Chapman et al., infra); (iv) open reading frames optimized forexpression in mammalian cells; and (v) a translational stop signalcodon.

TABLE 1A Exemplary Synthetic Env Expression Cassettes(SF162) SeqExpression Cassette Id Further Information gp120 SF162 30 wild-type;FIG. 16 gp140 SF162 31 wild-type; FIG. 17 gp160 SF162 32 wild-type; FIG.18 gp120.modSF162 33 none; FIG. 19 gp120.modSF162.delV2 34 deleted V2loop; FIG. 20 gp120.modSF162.delV1/V2 35 deleted V1 and V2; FIG. 21gp140.modSF162 36 none; FIG. 23 gp140.modSF162.delV2 37 deleted V2 loop;FIG. 24 gp140.modSF162.delV1/V2 38 deleted V1 and V2; FIG. 25gp140.mut.modSF162 39 mutated cleavage site; FIG. 26gp140.mut.modSF162.delV2 40 deleted V2; mutated cleavage site; FIG. 27gp140.mut.modSF162.delV1/V2 41 deleted V1 & V2; mutated cleavage site;FIG. 28 gp140.mut7.modSF162 42 mutated cleavage site; FIG. 29gp140.mut7.modSF162.delV2 43 mutated cleavage site; deleted V2; FIG. 30gp140.mut7.modSF162.delV1/V2 44 mutated cleavage site; deleted V1 andV2; FIG. 31 gp140.mut8.modSF162 45 mutated cleavage site; FIG. 32gp140.mut8.modSF162.delV2 46 mutated cleavage site; deleted V2; FIG. 33gp140.mut8.modSF162.delV1/V2 47 mutated cleavage site; deleted V1 andV2; FIG. 34 gp160.modSF162 48 none; FIG. 35 gp160.modSF162.delV2 49deleted V2 loop; FIG. 36 gp160.modSF162.delV1/V2 50 deleted V1 & V2;FIG. 37

TABLE 1B Exemplary Synthetic Env Expression Cassettes(US4) SeqExpression Cassette Id Further Information gp120 US4 51 wild-type; FIG.38 gp140 US4 52 wild-type; FIG. 39 gp160 US4 53 wild-type; FIG. 40gp120.modUS4 54 none; FIG. 41 gp120.modUS4.del 128-194 55 deletion in V1and V2 regions; FIG. 42 gp140.modUS4 56 none; FIG. 43 gp140.mut.modUS457 mutated cleavage site; FIG. 44 gp140TM.modUS4 58 native transmembraneregion; FIG. 45 gp140.modUS4.delV1/V2 59 deleted V1 and V2; FIG. 46gp140.modUS4.delV2 60 deleted V1; FIG. 47 gp140.mut.modUS4.delV1/V2 61mutated cleavage site; deleted V1 and V2; FIG. 48 gp140.modUS4.del128-194 62 deletion in V1 and V2 regions; FIG. 49 gp140.mut.modUS4.del128-194 63 mutated cleavage site; deletion in V1 and V2 regions; FIG. 50gp160.modUS4 64 none; FIG. 51 gp160.modUS4.delV1 65 deleted V1; FIG. 52gp160.modUS4.delV2 66 deleted V2; FIG. 53 gp160.modUS4.delV1/V2 67deleted V1 and V2; FIG. 54 gp160.modUS4del 128-194 68 deletion in V1 andV2 regions; FIG. 55

Alignments of the sequences presented in the above tables are presentedin FIGS. 66A and 66B.

A common region (Env-common) extends from nucleotide position 1186 tonucleotide position 1329 (SEQ ID NO:69, FIG. 56) relative to thewild-type US4 sequence and from nucleotide position 1117 to position1260 (SEQ ID NO:79, FIG. 57) relative to the wild-type SF162 sequence.The synthetic sequences of the present invention corresponding to theseregions are presented, as SEQ ID NO:71 (FIG. 58) for the synthetic EnvUS4 common region and as SEQ ID NO:72 (FIG. 59) for the synthetic EnvSF162 common region.

Percent identity to this sequence can be determined, for example, usingthe Smith-Waterman search algorithm (Time Logic, Incline Village, Nev.),with the following exemplary parameters: weight matrix=nuc4×4hb; gapopening penalty=20, gap extension penalty=5, reporting threshold=1;alignment threshold=20.

Various forms of the different embodiments of the present invention(e.g., constructs) may be combined.

F. Cloning Synthetic Env Expression Cassettes of the Present Invention.

The synthetic DNA fragments encoding the Env polypeptides were typicallycloned into the eucaryotic expression vectors described above for Gag,for example, pCMVKm2/pCMVlink (FIG. 4), pCMV6a, pESN2dhfr (FIG. 13A),pCMVIII (FIG. 13E; alternately designated as the pCMV-PL-E-dhfr/neovector).

Exemplary designations for pCMVlink vectors containing syntheticexpression cassettes of the present invention are as follows:pCMVlink.gp140.modSF162; pCMVlink.gp140.-modSF162.delV2;pCMVlink.gp140.mut.modSF162; pCMVlink.gp140.mut.modSF162.delV2;pCMVKm2.gp140modUS4; pCMVKm2.9p140.modUS4.delV2;pCMVKm2.gp140.mut.modUS4; and, pCMVKm2.gp140.mut.modUS4.delV1/V2.

G. Generation of Synthetic Tat Expression Cassettes

Tat coding sequences have also been modified according to the teachingsof the present specification. The wild type nucleotide sequence encodingtat from variant SF162 is presented in FIG. 76 (SEQ ID NO:85). Thecorresponding wild-type amino acid sequence is presented in FIG. 77 (SEQID NO:86). FIG. 81 (SEQ ID NO:89) shows the nucleotide sequence encodingthe amino terminal of the tat protein and the codon encoding cystein-22is underlined. Other exemplary constructs encoding synthetic tatpolypeptides are shown in FIGS. 78 and 79 (SEQ ID NOs:87 and 88). In oneembodiment (SEQ ID NO:88), the cystein residue at position 22 isreplaced by a glycine. Caputo et al. (1996) Gene Therapy 3:235 haveshown that this mutation affects the trans activation domain of Tat.

Various forms of the different embodiments of the invention, describedherein, may be combined.

H. Deposit of Vectors

Selected exemplary constructs shown below and described herein aredeposited at Chiron Corporation, Emeryville, Calif., 94662-8097, andwere sent to the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209 on Dec. 27, 1999.

Chiron Date Sent Plasmid Name Deposit # to ATCC pCMVgp160.modUS4 5094 27Dec 99 pCMVgp160delI.modUS4 5095 27 Dec 99 pCMVgp160del2.modUS4 5096 27Dec 99 pCMVgp160del-2.modUS4 5097 27 Dec 99 pCMVgp160del128-194.mod.US45098 27 Dec 99 pCMVgp140mut.modUS4del128-194 5100 27 Dec 99pCMVgp140.mut.mod.US 5101 27 Dec 99 pCMVgp160.modSF162 5125 27 Dec 99pCMVgp160.modSF162.delV2 5126 27 Dec 99 pCMVgp160.modSF162.delV1V2 512727 Dec 99 pCMVgp140.mut.modSF162delV2 5128 27 Dec 99pCMVgp140.mut7.modSF162 5129 27 Dec 99 pCMVgp140.mut7.modSF162delV2 513027 Dec 99 pCMVgp140.mut8.modSF162 5131 27 Dec 99pCMVgp140.mut8.modSF162delV2 5132 27 Dec 99pCMVgp140.mut8.modSF162delV1V2 5133 27 Dec 99pCMVKm2.Gagprot.Mod.SF2.GP1 5150 27 Dec 99 pCMVKm2.Gagprot.Mod.SF2.GP25151 27 Dec 99

Example 2 Expression Assays for the Synthetic Gag, Env and Tat CodingSequences A. Gag and Gag-Protease Coding Sequences

The HIV-1SF2 wild-type Gag (SEQ ID NO:1) and Gag-protease (SEQ ID NO:2)sequences were cloned into expression vectors having the same featuresas the vectors into which the synthetic Gag (SEQ ID NO:4) andGag-protease (SEQ ID NOs:5, 78 or 79)) sequences were cloned.

Expression efficiencies for various vectors carrying the HIV-15F2wild-type and synthetic Gag sequences were evaluated as follows. Cellsfrom several mammalian cell lines (293, RD, COS-7, and CHO; all obtainedfrom the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209) were transfected with 2 μg of DNA intransfection reagent LT1 (PanVera Corporation, 545 Science Dr., Madison,Wis.). The cells were incubated for 5 hours in reduced serum medium(Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The medium was then replacedwith normal medium as follows: 293 cells, IMDM, 10% fetal calf serum, 2%glutamine (BioWhittaker, Walkersville, Md.); RD and COS-7 cells, D-MEM,10% fetal calf serum, 2% glutamine (Opti-MEM, Gibco-BRL, Gaithersburg,Md.); and CHO cells, Ham's F-12, 10% fetal calf serum, 2% glutamine(Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The cells were incubated foreither 48 or 60 hours. Supernatants were harvested and filtered through0.45 μm syringe filters and, optionally, stored at −20° C.

Supernatants were evaluated using the Coulter p24-assay (CoulterCorporation, Hialeah, Fla., US), using 96-well plates coated with amurine monoclonal antibody directed against HIV core antigen. The HIV-1p24 antigen binds to the coated wells. Biotinylated antibodies againstHIV recognize the bound p24 antigen. Conjugated strepavidin-horseradishperoxidase reacts with the biotin. Color develops from the reaction ofperoxidase with TMB substrate. The reaction is terminated by addition of4NH₂SO₄. The intensity of the color is directly proportional to theamount of HIV p24 antigen in a sample.

The results of these expression assays are presented in Tables 2A and2B. Tables 2A and 2B shows data obtained using the syntheticGag-protease expression cassette of SEQ ID NO:5. Similar results wereobtained using the Gag-protease expression cassettes of SEQ ID NOs:78and 79.

Table 2: In Vitro Gag and Gagprot p24 Expression

TABLE 2a Increased in vitro expression from modified vs. native gagplasmids in supernatants and lysates from transiently transfected cellsnative (nat) ^(a) supernatant hours post total ng p24 exper- modified(sup) cell trans- (fold iment (mod) ^(b) lysate (lys) line fectionincrease) 1 nat sup 293 48 3.4 mod sup 293 48 1260 (371) nat sup 293 603.2 mod sup 293 60 2222 (694) 2 nat sup 293 60 1.8 mod sup 293 60 1740(966) 3 nat sup 293 60 1.8 mod sup 293 60  580 (322) 4 nat lys 293 601.5 mod lys 293 60  85 (57) 1 nat sup RD 48 5.6 mod sup RD 48  66 (12)nat sup RD 60 7.8 mod sup RD 60 70.2 (9)  2 nat lys RD 60 1.9 mod lys RD60 7.8 (4)  1 nat sup COS-7 48 0.4 mod sup COS-7 48 33.4 (84)  2 nat supCOS-7 48 0.4 mod sup COS-7 48  10 (25) nat lys COS-7 48 3  mod lys COS-748 14 (5) ^(a) pCMVLink.Gag.SF2.PRE ^(b) pCMVKm2.GagMod.SF2

TABLE 2B In vitro expression from modified gag and gagprotease plasmidsin supernatants and lysates from transiently transfected cellssupernatant (sup) hours post plasmid lysate (lys) cell line transfectiontotal ng p24^(d) Gag^(a) sup 293 60 760 GagProt(GP1)^(b) sup 293 60 380GagProt(GP2)^(c) sup 293 60 320 Gag lys 293 60 78 GagProt(GP1) lys 29360 1250 GagProt(GP2) lys 293 60 400 Gag sup COS-7 72 40 GagProt(GP1) supCOS-7 72 150 GagProt(GP2) sup COS-7 72 290 Gag lys COS-7 72 60GagProt(GP1) lys COS-7 72 63 GagProt(GP2) lys COS-7 72 58^(a)pCMVKM2.GagMod.SF2 ^(b)pCMVKM2.GagProtMod.SF2(GP1) gagprotease withcodon optimization and inactivation of INS in protease^(c)pCMVKm2.GagProtMod.SF2(GP2) gagprotease with only inactivation ofINS in protease ^(d)Shown are representative results from 3 independentexperiments for each cell line tested.

The data showed that the synthetic Gag and Gag-protease expressioncassettes provided dramatic increases in production of their proteinproducts, relative to the native (HIV-1SF2 wild-type) sequences, whenexpressed in a variety of cell lines.

B. Env Coding Sequences

The HIV-SF162 (“SF162”) wild-type Env (SEQ ID NO:1-3) and HIV-US4(“US4”) wild-type Env (SEQ ID NO:22-24) sequences were cloned intoexpression vectors having the same features as the vectors into whichthe synthetic Env sequences were cloned.

Expression efficiencies for various vectors carrying the SF162 and US4wild-type and synthetic Env sequences were evaluated essentially asdescribed above for Gag except that cell lysates were prepared in 40 μllysis buffer (1.0% NP40, 0.1 M Tris pH 7.5) and frozen at −20° C. andcapture ELISAs were performed as follows.

For Capture ELISAs, 250 ng of an ammonium sulfate IgG cut of goatpolyclonal antibody to gp120SF2/env2-3 was used to coat each well of a96-well plate (Corning, Corning, N. Y.). Serial dilutions of gp120/SF2protein (MID 167) were used to set the quantitation curve from whichexpression of US4 or SF162 gp120 proteins from transfection supernatantand lysates were calculated. Samples were screened undiluted and,optionally, by serial 2-fold dilutions. A human polyclonal antibody toHIV-1 gp120/SF2 was used to detect bound gp120 envelope protein,followed by horse-radish peroxidase (HRP)-labeled goat anti-human IgGconjugates. TME (Pierce, Rockford, Ill.) was used as the substrate andthe reaction is terminated by addition of 4N H₂SO₄. The reaction wasquantified by measuring the optical density (OD) at 450 nm. Theintensity of the color is directly proportional to the amount of HIVgp120 antigen in a sample. Purified SF2 gp120 protein was diluted andused as a standard.

The results of the transient expression assays are presented in Tables 3and 4. Table 3 depicts transient expression in 293 cells transfectedwith a pCMVKm2 vector carrying the Env cassette of interest. Table 4depicts transient expression in RD cells transfected with a pCMVKm2vector carrying the Env cassette of interest.

TABLE 3 Total Sup fold Total cell Cell lysate Total fold Native (N) Cellsup increase lysate fold increase Total increase Synthetic (S) Line (ng)(S v. N) (ng) (S v. N) (ng) (S v. N) N-gp120.US4 RD 87 <1 88S-gp120.modUS4 RD 690 8 2 5 693 8 N-gp140.US4 RD 526 0 526S-gp140.modUS4 RD 1305 2 1 2 1306 2 S-gp140mut.modUS4 RD 35 N/A 25 N/A60 N/A S-gp140TM.modUS4 RD 0 N/A 5 N/A 5 N/A N-gp160.US4 RD 0 8 8S-gp160.modUS4 RD 0 0 30 4 30 4

TABLE 4 CHO Cell Lines Expression Level of US4 Envelope Constructs MTXExpression Level* Constructs CHO Clone # Level (ng/ml) gp120.modUS4 13.2 μM 250-450 2 1.6 μM 350-450 3 200 nM 230-580 4 200 nM 300-500gp140.modUS4 1 1 μM 155-300 2 1 μM 100-260 3 1 μM 200-430 gp140.mut. 1 1μM 110-270 modUS4 2 1 μM 100-235 3 1 μM 100-220 gp140.modUS4. 1 50 nM313-587** delV1/V2 2 50 nM 237-667** 3 50 nM 492-527** gp140.mut. 1 50nM  46-328** modUS4.delV1/ 2 50 nM  82-318** V2 3 50 nM 204-385** *Allsamples measured at T-75 flask stage unless otherwise indicated **at 24well and 6 well plate stages ***in a three liter bioreactor perfusionculture this clone yielded approximately 2-5 μg/ml.

The data showed that the synthetic Env and expression cassettes provideda significant increase in production of their protein products, relativeto the native (HIV-1SF162 or US4 wild-type) sequences, when expressed ina variety of cell lines.

C. CHO Cell Line Env Expression Data

Chinese hamster ovary (CHO) cells were transfected with plasmid DNAencoding the synthetic HIV-1 gp120 or gp140 proteins (e.g., pESN2dhfr orpCMVIII vector backbone) using Mirus TransIT-LT1 polyamine transfectionreagent (Pan Vera) according to the manufacturers instructions andincubated for 96 hours. After 96 hours, media was changed to selectivemedia (F12 special with 250 μg/ml G418) and cells were split 1:5 andincubated for an additional 48 hours. Media was changed every 5-7 daysuntil colonies started forming at which time the colonies were picked,plated into 96 well plates and screened by gp120 Capture ELISA. Positiveclones were expanded in 24 well plates and screened several times forEnv protein production by Capture ELISA, as described above. Afterreaching confluency in 24 well plates, positive clones were expanded toT25 flasks (Corning, Corning, N. Y.). These were screened several timesafter confluency and positive clones were expanded to T75 flasks.

Positive T75 clones were frozen in LN2 and the highest expressing clonesamplified with 0-5 μM methotrexate (MTX) at several concentrations andplated in 100 mm culture dishes. Plates were screened for colonyformation and all positive closed were again expanded as describedabove. Clones were expanded an amplified and screened at each step bygp120 capture ELISA. Positive clones were frozen at each methotrexatelevel. Highest producing clones were grown in perfusion bioreactors (3L,100L) for expansion and adaptation to low serum suspension cultureconditions for scale-up to larger bioreactors.

Tables 5 and 6 show Capture ELISA data from CHO cells transfected withpCMVIII vector carrying a cassette encoding synthetic HIV-US4 and SF162Env polypeptides (e.g., mutated cleavage sites, modified codon usageand/or deleted hypervariable regions). Thus, stably transfected CHO celllines which express Env polypeptides (e.g., gp120, gp140-monomeric, andgp140-oligomeric) have been produced.

TABLE 5 CHO Cell Lines Expression Level of US4 Envelope ConstructsExpression MTX Level* Constructs CHO Clone # Level (ng/ml) gp120.modUS41 3.2 μM  250-450 2 1.6 μM  350-450 3 200 nM  230-580*** 4 200 nM 300-500 gp140.modUS4 1  1 μM 155-300 2  1 μM 100-260 3  1 μM 200-430gp140.mut.modUS4 1  1 μM 110-270 2  1 μM 100-235 3  1 μM 100-220gp140.modUS4.delV1/V2 1 50 nM 313-587** 2 50 nM 237-667** 3 50 nM492-527** gp140.mut.modUS4.delV1/V2 1 50 nM  46-328** 2 50 nM  82-318**3 50 nM 204-385** *All samples measured at T-75 flask stage unlessotherwise indicated **at 24 well and 6 well plate stages ***in a threeliter bioreactor perfusion culture this clone yielded approximately 2-5μg/ml.

TABLE 6 CHO Cell Lines Expression Level of SF162 Envelope ConstructsExpression MTX Level* Constructs CHO Clone # Level (ng/ml)gp120.modSF162 1  0  755-2705 2  0  928-1538 3  0  538-1609gp140.modSF162 1  20 nM 180-350 gp140.mut.modSF162 1  20 nM 164-451 2 20 nM 188-487 3  20 nM 233-804 gp120.modSF162.delV2 1 800 nM  528-15602 800 nM  487-1878 3 800 nM  589-1212 gp140.modSF162.delV2 1 800 nM300-600 2 800 nM 200-400 3 800 nM 200-500 gp140.mut.modSF162.delV2 1 800nM 300-700 2 400 nM 1161 3 800 nM 400-600 4 400 nM 1600-2176 *Allsamples measured at T-75 flask stage unless otherwise indicated

The results presented above demonstrate the ability of the constructs ofthe present invention to provide expression of Env polypeptides in CHOcells. Production of polypeptides using CHO cells provides (i) correctglycosylation patterns and protein conformation (as determined bybinding to panel of MAbs); (ii) correct binding to CD4 receptormolecules; (iii) absence of non-mammalian cell contaminants (e.g.,insect viruses and/or cells); and (iv) ease of purification.

D. Tat Coding Sequences

The HIV-SF162 (“SF162”) wild-type Tat (SEQ ID NO:85) sequences werecloned into expression vectors having the same features as the vectorsinto which the synthetic Tat sequences were cloned (SEQ ID NOs:87, 88and 89).

Expression efficiencies for various vectors carrying the SF162 wild-typeand synthetic Tat sequences are evaluated essentially as described abovefor Gag and Env using capture ELISAs with the appropriate anti-tatantibodies and/or CHO cell assays. Expression of the polypeptidesencoded by the synthetic cassettes is improved relative to wild type.

Example 3 Western Blot Analysis of Expression A. Gag and Gag-ProteaseCoding Sequences

Human 293 cells were transfected as described in Example 2 withpCMV6a-based vectors containing native or synthetic Gag expressioncassettes. Cells were cultivated for 60 hours post-transfection.Supernatants were prepared as described. Cell lysates were prepared asfollows. The cells were washed once with phosphate-buffered saline,lysed with detergent [1% NP40 (Sigma Chemical Co., St. Louis, Mo.) in0.1 M Tris-HCl, pH 7.5], and the lysate transferred into fresh tubes.SDS-polyacrylamide gels (pre-cast 8-16%; Novex, San Diego, Calif.) wereloaded with 20 μl of supernatant or 12.5 μl of cell lysate. A proteinstandard was also loaded (5 μl, broad size range standard; BioRadLaboratories, Hercules, Calif.). Electrophoresis was carried out and theproteins were transferred using a BioRad Transfer Chamber (BioRadLaboratories, Hercules, Calif.) to Immobilon P membranes (MilliporeCorp., Bedford, Mass.) using the transfer buffer recommended by themanufacturer (Millipore), where the transfer was performed at 100 voltsfor 90 minutes. The membranes were exposed to HIV-1-positive humanpatient serum and immunostained using o-phenylenediamine dihydrochloride(OPD; Sigma).

The results of the immunoblotting analysis showed that cells containingthe synthetic Gag expression cassette produced the expected p55 proteinat higher per-cell concentrations than cells containing the nativeexpression cassette. The Gag p55 protein was seen in both cell lysatesand supernatants. The levels of production were significantly higher incell supernatants for cells transfected with the synthetic Gagexpression cassette of the present invention. Experiments performed insupport of the present invention suggest that cells containing thesynthetic Gag-prot expression cassette produced the expected Gag-protprotein at comparably higher per-cell concentrations than cellscontaining the native expression cassette.

In addition, supernatants from the transfected 293 cells werefractionated on sucrose gradients. Aliquots of the supernatant weretransferred to Polyclear™ ultra-centrifuge tubes (Beckman Instruments,Columbia, Md.), under-laid with a solution of 20% (wt/wt) sucrose, andsubjected to 2 hours centrifugation at 28,000 rpm in a Beckman SW28rotor. The resulting pellet was suspended in PBS and layered onto a20-60% (wt/wt) sucrose gradient and subjected to 2 hours centrifugationat 40,000 rpm in a Beckman SW41ti rotor.

The gradient was then fractionated into approximately 10×1 ml aliquots(starting at the top, 20%-end, of the gradient). Samples were taken fromfractions 1-9 and were electrophoresed on 8-16% SDS polyacrylamide gels.Fraction number 4 (the peak fraction) corresponds to the expecteddensity of Gag protein VLPs. The supernatants from 293/synthetic Gagcells gave much stronger p55 bands than supernatants from 293/native Gagcells, and, as expected, the highest concentration of p55 in eithersupernatant was found in fraction 4.

These results demonstrate that the synthetic Gag expression cassetteprovides superior production of both p55 protein and VLPs, relative tothe native Gag coding sequences.

B. Env Coding Sequences

Human 293 cells were transfected as described in Example 2 withpCMVKm2-based; pCMVlink-based; p-CMVII-based or pESN2-based vectorscontaining native or synthetic Env expression cassettes. Cells werecultivated for 48 or 60 hours post-transfection. Cell lysates andsupernatants were prepared as described (Example 2). Briefly, the cellswere washed once with phosphate-buffered saline, lysed with detergent[1% NP40 (Sigma Chemical Co., St. Louis, Mo.)] in 0.1 M Tris-HCl, pH7.5], and the lysate transferred into fresh tubes. SDS-polyacrylamidegels (pre-cast 8-16%; Novex, San Diego, Calif.) were loaded with 20 μlof supernatant or 12.5 μl of cell lysate. A protein molecular weightstandard and an HIV SF2 gp120 positive control protein (5 μl, broad sizerange standard; BioRad Laboratories, Hercules, Calif.) were also loaded.Electrophoresis was carried out and the proteins were transferred usinga BioRad Transfer Chamber (BioRad Laboratories, Hercules, Calif.) toImmobilon P membranes (Millipore Corp., Bedford, Mass.) using thetransfer buffer recommended by the manufacturer (Millipore), where thetransfer was performed at 100 volts for 90 minutes. The membranes werethen reacted against polyclonal goat anti-gp120SF2/env2-3 anti-sera,followed by incubation with swine anti-goat IgG-peroxidase (POD) (Sigma,St. Louis, Mo.). Bands indicative of binding were visualized by addingDAB with hydrogen peroxide which deposits a brown precipitate on themembranes.

The results of the immunoblotting analysis showed that cells containingthe synthetic Env expression cassette produced the expected Env gpproteins of the predicted molecular weights as determined by mobilitiesin SDS-polyacrylamide gels at higher per-cell concentrations than cellscontaining the native expression cassette. The Env proteins were seen inboth cell lysates and supernatants. The levels of production weresignificantly higher in cell supernatants for cells transfected with thesynthetic Env expression cassette of the present invention.

C. Tat Coding Sequences

Human 293 cells are transfected as described in Example 2 with variousvectors containing native or synthetic Tat expression cassettes. Cellsare cultivated and isolated proteins analyzed as described above.Immunoblotting analysis shows that cells containing the synthetic Tatexpression cassette produced the expected Tat proteins of the predictedmolecular weights as determined by mobilities in SDS-polyacrylamide gelsat higher per-cell concentrations than cells containing the nativeexpression cassette.

Example 4 Purification of Env Polypeptides

A. Purification of Oligomeric gp140

Purification of oligomeric gp140 (o-gp140 US4) was conducted essentiallyas shown in FIG. 60. For the experiments described herein, o-gp140refers to oligomeric gp140 in either native or modified (e.g., optimizedexpression sequences, deleted, mutated, truncated, etc.) form. Briefly,concentrated (30-50×) supernatants obtained from CHO cell cultures wereloaded onto an anion exchange (DEAE) column which removed DNA and otherserum proteins. The eluted material was loaded onto a ceramichydroxyapatite column (CHAP) which bound serum proteins but not HIV Envproteins. The flow-through from the DEAE and CHAP columns was loadedonto a Protein A column as a precautionary step to remove any remainingserum immunoglobulins. The Env proteins in the flow-through were thencaptured using the lectin gluvanthus navalis (GNA, Vector Labs,Burlingame, Calif.). GNA has high affinity for mannose richcarbohydrates such as Env. The Env proteins were then eluted with GNAsubstrate. To remove other highly glycosylated proteins, a cationexchange column (SP) was used to purify gp140/gp120. In a final step,which separates gp120 from o-gp140, a gel filtration column was used toseparate oligomers from monomers. Sizing and chromatography analysis ofthe final product revealed that this strategy lead to the successfulisolation of oligomeric gp140.

B. Purification of gp120

Purification of gp120 was conducted essentially as previously describedfor other Env proteins. Briefly, concentrated supernatants obtained fromCHO cell cultures were loaded onto an anion exchange (DEAF) column whichremoved DNA and other serum proteins. The eluted material was loadedonto a ceramic hydroxyapatite column (CHAP) which bound serum proteinsbut not HIV Env proteins. The flow-through from the CHAP column wasloaded a cation exchange column (SP) where the flow-through wasdiscarded and the bound fraction eluted with salt. The elutedfraction(s) were loaded onto a Suprose 12/Superdex 200 Tandem column(Pharmacia-Upjohn, Uppsala, Sweden) from which purified gp120 wasobtained. Sizing and chromatography analysis of the final productrevealed that this strategy successfully purified gp120 proteins.

Example 5 Analysis of Purified Env Polypeptides

A. Analysis of o-gp140

It is well documented that HIV Env protein binds to CD4 only in itscorrect conformation. Accordingly, the ability of o-gp140 US4polypeptides, produced and purified as described above, to bind CD4cells was tested. O-gp140 US4 was incubated for 15 minutes withFITC-labeled CD4 at room temperature and loaded onto a Biosil 250(BioRad) size exclusion column using Waters HPLC. CD4-FITC has thelongest retention time (2.67 minutes), followed by CD4-FITC-gp120 (2.167min). The shortest retention time (1.9 min) was observed forCD4-FITC-o-gp140 US4 indicating that, as expected, o-gp140 US4 binds toCD4 forming a large complex which reduces retention time on the column.Thus, the o-gp140 US4 produced and purified as described above is of thecorrect size and conformation.

In addition, the US4 o-gp140, purified as described above, was alsotested for its ability to bind to a variety of monoclonal antibodieswith known epitope specificities for the CD4 binding site, the CD4inducible site, the V3 loop and oligomer-specific gp41 epitope. O-gp140bound strongly to these antibodies, indicating that the purified proteinretains its structural integrity.

B. Analysis of gp120

As described above, CD4-FITC binds gp120, as demonstrated by thedecreased retention time on the HPLC column. Thus, US4 gp120 purified bythe above method retains its conformational integrity. In addition, theproperties of purified gp120 can be tested by examining its integrityand identity on western blots, as well as, by examining proteinconcentration, pH, conductivity, endotoxin levels, bioburden and thelike. US4 gp120, purified as described above, was also tested for itsability to bind to a variety of monoclonal antibodies with known epitopespecificities for the CD4 binding site, the CD4 inducible site, the V3loop and oligomer-specific gp41 epitope. The pattern of mAb binding togp120 indicated that the purified protein retained its structuralintegrity, for example, the purified gp120 did not bind the mAb havingthe oligomer-specific gp41 epitope (as expected).

Example 6 Electron Microscopic Evaluation of VLP Production

The cells for electron microscopy were plated at a density of 50-70%confluence, one day before transfection. The cells were transfected with10 μg of DNA using transfection reagent LT1 (Panvera) and incubated for5 hours in serum-reduced medium (see Example 2). The medium was thenreplaced with normal medium (see Example 2) and the cells were incubatedfor 14 hours (COS-7) or 40 hours (CHO). After incubation the cells werewashed twice with PBS and fixed with 2% glutaraldehyde. Electronmicroscopy was performed by Prof. T. S. Benedict Yen, Veterans Affairs,Medical Center, San Francisco, Calif.).

Electron microscopy was carried out using a transmission electronmicroscope (Zeiss 10c). The cells were pre-stained with osmium andstained with uranium acetate and lead citrate. The magnification was100,000×.

FIGS. 3A and 3B show micrographs of CHO cells transfected with pCMVKM2carrying the synthetic Gag expression cassette (SEQ ID NO:5) or carryingthe Gag-prot expression cassette (SEQ ID NO:79). In the figure, free andbudding immature virus-like-particles (VLP) of the expected size (100nm) are seen for the Gag expression cassette (FIG. 3A) and both immatureand mature VLPs are seen for the Gag-prot expression cassette (FIG. 38).COS-7 cells transfected with the same vector have the same expressionpattern. VLP can also be found intracellularly in CHO and COS-7 cells.

Native and synthetic Gag expression cassettes were compared for theirassociated levels of VLP production when used to transfect human 293cells. The comparison was performed by density gradientultracentrifugation of cell supernatants and Western-blot analysis ofthe gradient fractions. There was a clear improvement in production ofVLPs when using the synthetic Gag construct.

Example 7 Expression of Virus-Like Particles in the Baculovirus SystemA. Expression of Native HIV p55 Gag

To construct the native HIV p55 Gag baculovirus shuttle vector, theprototype SF2 HIV p55 plasmid, pTM1-Gag (Selby M. J., et al., J. Virol.71(10):7827-7831, 1997), was digested with restriction endonucleasesNcoI and BamHI to extract a 1.5 Kb fragment that was subsequentlysubcloned into pAcC4 (Bio/Technology 6:47-55, 1988), a derivative ofpAc436. Generation of the recombinant baculovirus was achieved byco-transfecting 2 μg of the HIV p55 Gag pAcC4 shuttle vector with 0.5 μgof linearized, Autographa californica baculovirus (AcNPV) wild-typeviral DNA into Spodoptera frugiperda (Sf9) cells (Kitts, P. A., Ayres M.D., and Possee R. D., Nucleic Acids Res. 18:5667-5672, 1990). Theisolation of recombinant virus expressing HIV p55 Gag was performedaccording to standard techniques (O'Reilly, D. R., L. K. Miller, and V.A. Luckow, Baculovirus Expression Vector: A Laboratory Manual, W.H.Freeman and Company, New York, 1992).

Expression of the HIV p55 Gag was achieved using a 500 ml suspensionculture of, Sf9 cells grown in serum-free medium (Miaorella, B., D.Inlow, A. Shauger, and D. Harano, Bio/Technology 6:1506-1510, 1988) thathad been infected with the HIV p55 Gag recombinant baculovirus at amultiplicity of infection (MOI) of 10. Forty-eight hours post-infection,the supernatant was separated by centrifugation and filtered through a0.2 μm filter. Aliquots of the supernatant were then transferred toPolyclear™ (Beckman Instruments, Palo Alto, Calif.) ultracentrifugetubes, underlaid with 20% (wt/wt) sucrose, and subjected to 2 hourscentrifugation at 24,00 rpm using a Beckman SW28 rotor.

The resulting pellet was suspended in Tris buffer (20 mM Tris HCl, pH7.5, 250 mM NaCl, and 2.5 mM ethylenediaminetetraacetic acid [EDTA]),layered onto a 20-60% (wt/wt) sucrose gradient, and subjected to 2 hourscentrifugation at 40,000 rpm using a Beckman SW41ti rotor. The gradientwas then fractionated starting at the top (20% sucrose) of the gradientinto approximately twelve 0.75 ml aliquots. A sample of each fractionwas electrophoresed on 8-16% SDS polyacrylamide gels and the resultingbands were visualized after commassie staining (FIG. 4). Additionalaliquots were subjected to refractive index analysis.

The results shown in FIG. 4 indicated that the p55 Gag virus-likeparticles banded at a sucrose density of range of 1.15-1.19 g/ml withthe peak at approximately 1.17 g/ml. The peak fractions were pooled andconcentrated by a second 20% sucrose pelleting. The resulting pellet wassuspended in 1 ml of Tris buffer (described above). The total proteinyield as estimated by Bicimchrominic Acid (BCA) (Pierce Chemical,Rockford, Ill.) was 1.6 mg.

B. Expression of Synthetic HIV p55 Gag

A baculovirus shuttle vector containing the synthetic p55 Gag sequencewas constructed as follows. The synthetic HIV p55 expression cassette(Example 1) was digested with restriction enzyme Sail followed byincubation with T4-DNA polymerase. The resulting fragment was isolated(PCR Clean-Up™, Promega, Madison, Wis.) and then digested with BamHIendonuclease. The shuttle vector pAcC13 (Munemitsu S., et al., Mol CellBiol. 10(11):5977-5982, 1990) was linearized by digestion with EcoI,followed by incubation with T4-DNA polymerase, and then isolated (PCRClean-Up™). The linearized vector was digested with BamHI, treated withalkaline phosphatase, and isolated by size fragmentation in an agarosegel. The isolated 1.5 kb fragment was ligated with the prepared pAcC13vector. The resulting clone was designated pAcC13-Modif.p55Gag.

The expression conditions for the synthetic HIV p55 VLPs differed fromthose of the native p55 Gag as follows: a culture volume of 1 liter usedinstead of 500 ml; Trichoplusia ni (Tn5) (Wickham, T. J., and Nermerow,G. R., BioTechnology Progress, 9:25-30, 1993) insect cells were usedinstead of Sf9 insect cells; and, an MOI of 3 was instead of an MOI of10. Experiments performed in support of the present invention showedthat there was no appreciable difference in expression level between theSf9 and Tn5 insect cells with the native p55 clone. In terms of MOI,experience with the native p55 clone suggested that an MOI of 10resulted in higher expression (approximately 2-fold) of VLPs than alower MOI.

The sucrose pelleting and banding methods used for the synthetic p55VLPs were similar to those employed for the native p55 VLPs (describedabove), with the following exceptions: pelleted VLPs were suspended in 4ml of phosphate buffered saline (PBS) instead of 1.0 ml of the Trisbuffer; and four, 20-60% sucrose gradients were used instead of a singlegradient. Also, due to the high concentration of banded VLPs, furtherconcentration by pelleting was not required. The peak fractions from all4 gradients were simply dialyzed against PBS. The approximate density ofthe banded VLPs ranged from 1.23-1.28 g/ml. A total protein yield asestimated by BCA was 46 mg. Results from the sucrose gradient banding ofthe synthetic p55 are shown in FIG. 5.

A comparison of the total amount of purified HIV p55 Gag from severalpreparations obtained from the two baculovirus expression cassettes hasbeen summarized in FIG. 6. The average yield from the native p55 was3.16 mg/liter of culture (n=5, standard deviation (sd) ±1.07,range=1.8-4.8 mg/L) whereas the average yield from the synthetic p55 wasmore than ten-fold higher at 44.5 mg/liter of culture (n=2, sd=±6.4).

In addition to a higher total protein yield, the final product from thesynthetic p55-expressed Gag consistently contained lower amounts ofcontaminating baculovirus proteins than the final product from thenative p55-expressed Gag. This difference can be seen in the twocommassie-stained gels FIGS. 4 and 5.

C. Expression of Native and Synthetic Gag-Core

Expression of the HIV p55 Gag/HCV Core 173 (SEQ ID NO:8) was achievedusing a 2.5 liter suspension culture of Sf9 cells grown in serum-freemedium (Miaorella, B., D. Inlow, A. Shauger, and D. Harano. 1988Bio/Technology 6:1506-1510). The cells were infected with an HIV p55Gag/HCV Core 173 recombinant baculovirus. Forty-eight hourspost-infection, the supernatant was separated from the cells bycentrifugation and filtered through a 0.2 μm filter. Aliquots of thesupernatant were then transferred to a Polyclear™ (Beckman Instruments,Palo Alto, Calif.) ultracentrifuge tubes containing 30% (wt/wt) sucrose,and subjected to 2 hours of centrifugation at 24,000 rpm in a BeckmanSW28 rotor and ultracentrifuge.

The resulting pellet was suspended in Tris buffer (50 mM Tris-HCl, pH7.5, 500 mM NaCl) and layered onto a 30-60% (wt/wt) sucrose gradient andsubjected to 2 hours centrifugation at 40,000 rpm in a Beckman SW41tirotor and ultracentrifuge. The gradient was then fractionated startingat the top (30%) of the gradient into approximately 11×1.0 ml aliquots.A sample of each fraction was electrophoresed on 8-16% SDSpolyacrylamide gels and the resulting bands were visualized aftercommassie staining.

A subset of aliquots were also subjected to Western blot analysis usingmonoclonal antibody 76C.5EG (Steimer, K. S., et al., Virology150:283-290, 1986) which is specific for HIV p24 (a subunit of HIV p55).The peak fractions from the sucrose gradient were pooled andconcentrated by a second 20% sucrose pelleting. The resulting pellet wassuspended in 1 ml of buffer Tris buffer and the total protein yield asestimated by BCA (Pierce Chemical, Rockford, Ill.) was ˜1.0 mg.

The results from the SDS PAGE are shown in FIG. 8 and the anti-p24Western blot results are shown in FIG. 9. Taken together, these resultsindicate that the HIV p55 Gag/HCV Core 173 chimeric VLPs banded at asucrose density similar to that of the HIV p55 Gag VLPs and the visibleprotein band that migrated at a molecular weight of ˜72,000 kd wasreactive with the HIV p24-specific monoclonal antibody. An additionalimmunoreactive band at approximately 55,000 kd also appeared to bereactive with the anti-p24 antibody and may be a degradation product.

Although aliquots from the above preparation were not tested forreactivity with an HCV Core-specific antibody (an anti-CD22 rabbitserum), results from a similar preparation are shown in FIG. 10 andindicate that the main HCV Core-specific reactivity migrates at anapproximate molecular weight of 72,000 kd which is in accordance withthe predicted molecular weight of the chimeric protein.

The expression conditions for the synthetic HIV p55 Gag/HCV Core 173(SEQ ID NO:8) VLPs differed from those of the native p55 Gag and are asfollows: a culture volume of 1 liter used instead of 2.5 liters,Trichoplusia ni (Tn5) (Wickham, T. J., and Nemerow, G. R. 1993BioTechnology Progress, 9:25-30) insect cells were used instead of Sf9insect cells and an MOI of 3 was instead of an MOI of 10. The sucrosepelleting and banding methods used for the synthetic HIV p55 Gag/HCVCore 173 VLPs were similar to those employed for the native HIV p55Gag/HCV Core 173 VLPs. However, differences included: pelleted VLPs weresuspended in 1 ml of phosphate buffered saline (PBS) instead of 1.0 mlof the Tris buffer, and a single 20-60% sucrose gradients was used. Acomparison of the total amount of purified HIV p55 Gag/HCV Core 173 frommultiple preparations obtained from the two baculovirus expressioncassettes showed that there was an increase in expression using thesynthetic HIV p55 Gag/HCV Core 173 cassette.

D. Alternative Method for the Enrichment of HIV p55 Gag VLPs

In addition to purification from the media, p55 (Gag protein) expressedin baculovirus (e.g., using a synthetic expression cassette of thepresent invention) can also be purified as virus-like particles from theinfected insect cells. For example, forty-eight hours post infection,the media and cell pellet are separated by centrifugation and the cellpellet is stored at −70° C. until future use. At the time of processing,the cell pellet is suspended in 5 volumes of hypotonic lysis buffer (20mM Tris-HCl, pH 8.2, 1 mM EGTA; 1 mM MgCl, and Complete ProteaseInhibitor® (Boehringer Mannheim Corp., Indianapolis, Ind.]). If needed,the cells are then dounced 8-10 times to complete cell lysis.

The lysate is then centrifuged at approximately 1000-1500×g for 20minutes. The supernatant is decanted into UltraClear™ tubes, underlayedwith 20% sucrose (w/w) and centrifuged at 24,000 rpm in SW28 buckets for2 hours. The resulting pellet is suspended in Tris buffer (20 mM TrisHCl, pH 7.5, 250 mM NaCl, and 2.5 mM ethylene-diamine-tetraacetic acid(EDTA) with 0.1% IGEPAL detergent (Sigma Chemical, St. Louis, Mo.) and250 units/ml of benzonase (American International Chemical, Inc.,Natick, Mass.) and incubated at 4° C. for at least 30 minutes. Thesuspension is subsequently layered onto a 20-60% sucrose gradient andspun at 40,000 rpm using an SW41ti rotor for 20-24 hours.

After ultracentrifugation, the sucrose gradient is fractionated andaliquots run on SDS PAGE to identify peak fractions. The peak fractionsare dialyzed against PBS and measured for protein content. Negativelystained electron mircographs typically show non-enveloped VLPs somewhatsmaller in diameter (80-120 nm) than the budded VLPs. HIV Gag VLPsprepared in this manner are also capable of generating Gag-specific CTLresponses in mice.

Example 8 In Vivo Immunogenicity of Synthetic Gag Expression CassettesA. Immunization

To evaluate the possibly improved immunogenicity of the synthetic Gagexpression cassettes, a mouse study was performed. The plasmid DNA,pCMVKM2 carrying the synthetic Gag expression cassette, was diluted tothe following final concentrations in a total injection volume of 100μl: 20 μg, 2 μg, 0.2 μg, and 0.02 μg. To overcome possible negativedilution effects of the diluted DNA, the total DNA concentration in eachsample was brought up to 20 μg using the vector (pCMVKM2) alone. As acontrol, plasmid DNA of the native Gag expression cassette was handledin the same manner. Twelve groups of four Balb/c mice (Charles River,Boston, Mass.) were intramuscularly immunized (50 μl per leg,intramuscular injection into the tibialis anterior) according to theschedule in Table 7.

TABLE 7 Concentration Gag Expression of Gag plasmid Immunized at GroupCassette DNA (μg) time (weeks): 1 Synthetic 20 0¹, 4  2 Synthetic 2 0, 43 Synthetic 0.2 0, 4 4 Synthetic 0.02 0, 4 5 Synthetic 20 0 6 Synthetic2 0 7 Synthetic 0.2 0 8 Synthetic 0.02 0 9 Native 20 0 10 Native 2 0 11Native 0.2 0 12 Native 0.02 0 ¹= initial immunization at “week 0” Groups1-4 were bled at week 0 (before immunization), week 4, week 6, week 8,and week 12. Groups 5-12 were bled at week 0 (before immunization) andat week 4.

B. Humoral Immune Response

The humoral immune response was checked with an anti-HIV Gag antibodyELISAs (enzyme-linked immunosorbent assays) of the mice sera 0 and 4weeks post immunization (groups 5-12) and, in addition, 6 and 8 weekspost immunization, respectively, 2 and 4 weeks post second immunization(groups 1-4).

The antibody titers of the sera were determined by anti-Gag antibodyELISA. Briefly, sera from immunized mice were screened for antibodiesdirected against the HIV p55 Gag protein. ELISA microtiter plates werecoated with 0.2 μg of HIV-1_(SF2) p24-Gag protein per well overnight andwashed four times; subsequently, blocking was done with PBS-0.2% Tween(Sigma) for 2 hours. After removal of the blocking solution, 100 μl ofdiluted mouse serum was added. Sera were tested at 1/25 dilutions and byserial 3-fold dilutions, thereafter. Microtiter plates were washed fourtimes and incubated with a secondary, peroxidase-coupled anti-mouse IgGantibody (Pierce, Rockford, Ill.). ELISA plates were washed and 100 μlof 3,3′,5,5′-tetramethyl benzidine (TMB; Pierce) was added per well. Theoptical density of each well was measured after 15 minutes. The titersreported are the reciprocal of the dilution of serum that gave ahalf-maximum optical density (O. D.). The ELISA results are presented inTable 8.

TABLE 8 Inoculum Expression Sera - Sera - Sera - Group (μg) cassetteWeek 4 ³ Week 6 Week 8 1 20 S ¹ - gag 98 455 551 2 2 S - gag 59 1408 2273 0. S - gag 29 186 61 4 0.02 S - gag <20 <20 <20 5 20 S - gag 67 n.a. ⁴n.a. 6 2 S - gag 63 n.a. n.a. 7 0. S - gag 57 n.a. n.a. 8 0.02 S - gag<20 n.a. n.a. 9 20 N ² - gag 43 n.a. n.a. 10 2 N - gag <20 n.a. n.a. 110. N - gag <20 n.a. n.a. 12 0.02 N - gag <20 n.a. n.a. ¹ = synthetic gagexpression cassette (SEQ ID NO: 4) ² = native gag expression cassette(SEQ ID NO: 1) ³ = geometric mean antibody titer ⁴ = not applicable

The results of the mouse immunizations with plasmid-DNAs show that thesynthetic expression cassettes provide a clear improvement ofimmunogenicity relative to the native expression cassettes. Also, thesecond boost immunization induced a secondary immune response after twoweeks (groups 1-3).

C. Cellular Immune Response

The frequency of specific cytotoxic T-lymphocytes (CTL) was evaluated bya standard chromium release assay of peptide pulsed Balb/c mouse CD4cells. Gag expressing vaccinia virus infected CD-8 cells were used as apositive control (vvGag). Briefly, spleen cells (Effector cells, E) wereobtained from the BALB/c mice immunized, as described above (Table 8)were cultured, restimulated, and assayed for CTL activity against Gagpeptide-pulsed target cells as described (Doe, B., and Walker, C. M.,AIDS 10(7):793-794, 1996). The HIV-1_(SF2) Gag peptide used was p7g SEQID NO:10. Cytotoxic activity was measured in a standard ⁵¹Cr releaseassay. Target (T) cells were cultured with effector (E) cells at variousE:T ratios for 4 hours and the average cpm from duplicate wells was usedto calculate percent specific ⁵¹Cr release. The results are presented inTable 9.

Cytotoxic T-cell (CTL) activity was measured in splenocytes recoveredfrom the mice immunized with HIV Gag DNA (compare Effector column, Table9, to immunization schedule, Table 8). Effector cells from the GagDNA-immunized animals exhibited specific lysis of Gag p7g peptide-pulsedSV-BALB (MHC matched) targets cells indicative of a CTL response. Targetcells that were peptide-pulsed and derived from an MHC-unmatched mousestrain (MC57) were not lysed (Table 9; MC/p7g).

TABLE 9 Cytotoxic T-lymphocyte (CTL) responses in mice immunized withHIV-1 gag DNA

*representative results of two animals per DNA-dose; positive CTLresponses are indicated by boxed data

The results of the CTL assays show increased potency of synthetic Gagexpression cassettes for induction of cytotoxic T-lymphocyte (CTL)responses by DNA immunization.

Example 9 In Vivo Immunization with Env Polypeptides

A. Immunogenicity Study of US4 o-gp140 in Ras-3c Adjuvant System

Studies have been conducted using rabbits immunized with US4 o-gp140purified as described above. Studies are also underway in animals todetermine immunogenicity of US4 gp120, SF162 o-gp140 and SF162 gp120.

Two rabbits (#1 and #2) were immunized intramuscularly at 0, 4, 12 and24 weeks with 50 μg of US4 o-gp140 in the Ribi™ adjuvant system(RAS-3c), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% Tween 80, and one or more bacterial cell wall components from thegroup consisting of monophosphorylipid A (MPL, Ribi Immunochem,Hamilton, Mont.). In each experiment described herein, o-gp140 can benative, mutated and/or modified. Antibody responses directed against theUS4 o-gp140 protein were measured by ELISA. Results are shown in Table10.

TABLE 10 Approximate o-gp140 ELISA Rabbit/sample titer pre-immunization0 #1: post1 (0 week immuniz) 400 #1: post2 (4 week immuniz) 15,000 #1:post3 (12 week immuniz) 50,000 #1: post4 (24 week immuiz) 100,000 #2:post1 (0 week immuniz) 600 #2: post2 (4 week immuniz) 12,000 #2: post3(12 week immuniz) 25,000 #2: post4 (24 week immuiz) 55,000

The avidities of antibodies directed against the US4 o-gp140 proteinwere measured in a similar ELISA format employing successive washes withincreasing concentrations of ammonium isothiocynate. Results are shownin Table 11.

TABLE 11 Approx. Antibody avidity (NH₄HCN Time of sample Conc. in M)pre-immunization 0.02 post1 (0 week immuniz) 1.8 post2 (4 week immuniz)3.5 post3 (12 week immuniz) 5.5 post4 (24 week immuniz) 5.1

These results show that US4 o-gp140 is highly immunogenic and able toinduce substantial antibody responses after only one or twoimmunizations.

B. Immunogenicity of US4 o-gp140 in MF59-Based Adjuvants

Groups of 4 rabbits were immunized intramuscularly at 0, 4, 12 and 24weeks with various doses of US4 o-gp140 protein in three differentMF59-based adjuvants (MF59 is described in International Publication No.WO 90/14837 and typically contains 5% Squalene, 0.5% Tween 80, and 0.5%Span 85). Antibody titers were measured post-third by ELISA using SF2gp120 to coat the plates. QHC is a quill-based adjuvant (Iscotek,Uppsala, Sweden). Results are shown in Table 12.

TABLE 12 Antigen dose (μg) Adjuvant Anti-gp120_(SF2) Ab GMT* 12.5 MF597231 25 MF59 8896 50 MF59 12822 12.5 MF59/MPL 24146 25 MF59/MPL 27199 50MF59/MPL 23059 50 MF59/MPL/QHC 31759 *GMT = geometric mean titer

Thus, adjuvanted o-gp140 generated antigen-specific antibodies. Further,the antibodies were shown to increased in avidity over time.

C. Neutralizing Antibodies

Neutralizing antibodies post-third immunization were measured againstHIV-1 SF2 in a T-cell line adapted virus (TCLA) assay and againstPBMC-grown HIV-1 variants SF2, SF162 and 119 using the CCR5+ CEMx174LTR-GFP reporter cell line, 5.25 (provided by N. Landau, Salk Institute,San Diego, Calif.) as target cells. Results are shown in Table 13.

TABLE 13 Neutralizing antibody responses in rabbits immunized witho-gp140.modUS4 protein SF2 SF2 SF162 119 Group Animal TCLA* PBMC^(#)PBMC^(#) PBMC^(#) Experiment 1 o-gp140/ 217 >640 100% 49 17 Ras-3c218 >640 96 37 29 50 mg Experiment 2 o-gp140/ 792 45 71 39 26 MF59 79350 87 26 4 50 mg 794 59 87 13 0 795 128 92 15 0 o-gp140/ 804 173 91 4718 MF59 + MPL 805 134 93 28 4 50 mg 806 N.D.** 95 49 13 807 441 100 3115 o-gp140/MF59 + 808 465 98 46 40 MPL + QHC 809 496 100 44 39 50 mg810 >640 101 27 4 811 92 92 24 37 *TCLA neutralizing antibody titers(50% inhibition). **Not Determined ^(#)% Inhibition at 1:10 dilution ofsera with any detectable non-specific inhibition in pre-bleedssubtracted.

The above studies in rabbits indicate that the US4 o-gp140 protein ishighly immunogenic. When administered with adjuvant, this protein wasable to induce substantial antibody responses after only one or twoimmunizations. Moreover, the adjuvanted o-gp140 protein was able togenerate antigen-specific antibodies which increased in avidity aftersuccessive immunizations, and substantial neutralizing activity againstT-cell line adapted HIV-1.Neutralizing activity was also observedagainst PBMC-grown primary HIV strains, including the difficult toneutralize CCR5 co-receptor (R5)-utilizing isolates, SF162 and 119.

Example 10 In Vivo Immunogenicity of Synthetic Env Expression CassettesA. General Immunization Methods

To evaluate the immunogenicity of the synthetic Env expressioncassettes, studies using guinea pigs, rabbits, mice, rhesus macaques andbaboons were performed. The studies were structured as follows: DNAimmunization alone (single or multiple); DNA immunization followed byprotein immunization (boost); DNA immunization followed by Sindbisparticle immunization; immunization by Sindbis particles alone.

B. Humoral Immune Response

The humoral immune response was checked in serum specimens fromimmunized animals with an anti-HIV Env antibody ELISAs (enzyme-linkedimmunosorbent assays) at various times post-immunization. The antibodytiters of the sera were determined by anti-Env antibody ELISA asdescribed above. Briefly, sera from immunized animals were screened forantibodies directed against the HIV gp120 or gp140 Env protein. Wells ofELISA microtiter plates were coated overnight with the selected Envprotein and washed four times; subsequently, blocking was done withPBS-0.2% Tween (Sigma) for 2 hours. After removal of the blockingsolution, 100 μl of diluted mouse serum was added. Sera were tested at1/25 dilutions and by serial 3-fold dilutions, thereafter. Microtiterplates were washed four times and incubated with a secondary,peroxidase-coupled anti-mouse IgG antibody (Pierce, Rockford, Ill.).ELISA plates were washed and 100 μl of 3,3′, 5,5′-tetramethyl benzidine(TMB; Pierce) was added per well. The optical density of each well wasmeasured after 15 minutes. Titers are typically reported as thereciprocal of the dilution of serum that gave a half-maximum opticaldensity (O.D.).

Example 11 DNA-immunization of Baboons Using Synthetic Gag ExpressionCassettes

A. Baboons

Four baboons were immunized 3 times (weeks 0, 4 and 8) bilaterally,intramuscular into the quadriceps using 1 mg pCMVKM2.GagMod.SF2plasmid-DNA (Example 1). The animals were bled two weeks after eachimmunization and a p24 antibody ELISA was performed with isolatedplasma. The ELISA was performed essentially as described in Example 5except the second antibody-conjugate was an anti-human IgG, g-chainspecific, peroxidase conjugate (Sigma Chemical Co., St. Louis, Md.63178) used at a dilution of 1:500. Fifty μg/ml yeast extract was addedto the dilutions of plasma samples and antibody conjugate to reducenon-specific background due to preexisting yeast antibodies in thebaboons. The antibody titer results are presented in Table 14.

TABLE 14 Immunization no. Weeks Antigen wpi^(a)/Baboon No. Ab-titer^(b)1 0 gagmod 0 w/219 <10 DNA 0 w/220 <10 0 w/221 <10 0 w/222 <10 6 2 wp1st/219 <10 2 wp 1st/220 <10 2 wp 1st/221 <10 2 wp 1st/222 15 4 14gagmod 2 wp 4th/219 <10 DNA 2 wp 4th/220 88 2 wp 4th/221 <10 2 wp4th/222 56 5 30 gagmod 2 wp 5th/219 <10 DNA 2 wp 5th/220 391 2 wp5th/221 237 2 wp 5th/222 222 6 46 gag VLP 2 wp 6th/219 753 protein 2 wp6th/219 4330 2 wp 6th/219 5000 2 wp 6th/219 2881 ^(a)wpi = weeks postimmunization ^(b)geometric mean antibody titer

In Table 14, pre-bleed data are given as Immunization No. 0; data forbleeds taken 2 weeks post-first immunization are given as ImmunizationNo. 1; data for bleeds taken 2 weeks post-second immunization are givenas Immunization No. 2; and, data for bleeds taken 2 weeks post-thirdimmunization are given as Immunization No. 3.

Further, lymphoproliferative responses to p24 antigen were also observedin baboons 221 and 222 two weeks post-fourth immunization (at week 14),and enhanced substantially post-boosting with VLP (at week 44 and 76).Such proliferation results are indicative of induction of T-helper cellfunctions.

B. Rhesus Macaques

The improved potency of the codon-modified gag expression plasmidobserved in mouse and baboon studies was confirmed in rhesus macaques.Four of four macaques had detectable Gag-specific CTL after two or three1 mg doses of modified gag plasmid. In contrast, in a previous study,only one of four macaques given 1 mg doses of plasmid-DNA encoding thewild-type HIV-1_(SF2) Gag showed strong CTL activity that was notapparent until after the seventh immunization. Further evidence of thepotency of the modified gag plasmid was the observation that CTL fromtwo of the four rhesus macaques reacted with three nonoverlapping Gagpeptide pools, suggesting that as many as three different Gag peptidesare recognized and indicating that the CTL response is polyclonal.Additional quantification and specificity studies are in progress tofurther characterize the T cell responses to Gag in theplasmid-immunized rhesus macaques. DNA immunization of macaques with themodified gag plasmid did not result in significant antibody responses,with only two of four animals seroconverting at low titers. In contrast,in the same study the majority of macaques in groups immunized withp55Gag protein seroconverted and had strong Gag-specific antibodytiters. These data suggest that a prime-boost strategy (DNA-prime andprotein-boost) could be very promising for the induction of a strong CTLand antibody response.

In sum, these results demonstrate that the synthetic Gag plasmid DNA isimmunogenic in non-human primates. When similar experiments were carriedout using wild-type Gag plasmid DNA no such induction of anti-p24antibodies was observed after four immunizations.

Example 12 DNA- and Protein Immunizations of Animals Using EnvExpression Cassettes and Polypeptides A. Guinea Pigs

Groups comprising six guinea pigs each were immunized intramuscularly at0, 4, and 12 weeks with plasmid DNAs encoding the gp120.modUS4,gp140.modUS4, gp140.modUS4.delV1, gp140.modUS4.delV2,gp140.modUS4.delV1/V2, or gp160.modUS4 coding sequences of theUS4-derived Env. The animals were subsequently boosted at 18 weeks witha single intramuscular dose of US4 o-gp140.mut.modUS4 protein in MF59adjuvant. Anti-gp120 SF2 antibody titers (geometric mean titers) weremeasured at two weeks following the third DNA immunization and at twoweeks after the protein boost. Results are shown in Table 15.

TABLE 15 GMT post-DNA GMT post-protein Group immuniz. boost gp120.modUS42098 9489 gp140.modUS4 190 5340 gp140.modUS4.delV1 341 7808gp140.modUS4.delV2 386 8165 gp140.modUS4.delV1/V2 664 8270 gp160.modUS4235 9928

These results demonstrate the usefulness of the synthetic constructs togenerate immune responses, as well as, the advantage of providing aprotein boost to enhance the immune response following DNA immunization.

B. Rabbits

Rabbits were immunized intramuscularly and intradermally using a Biojectneedless syringe with plasmid DNAs encoding the following syntheticSF162 Env polypeptides: gp120.modSF162, gp120.modSF162.delV2,gp140.modSF162, gp140.modSF162.delV2, gp140.mut.modSF162,gp140.mut.modSF162.delV2, gp160.modSF162, and gp160.modSF162.delV2.Approximately 1 mg of plasmid DNA (pCMVlink) carrying the synthetic Envexpression cassette was used to immunize the rabbits. Rabbits wereimmunized with plasmid DNA at 0, 4, and 12 weeks. At two weeks after thethird immunization all of the constructs were shown to have generatedsignificant antibody titers in the test animals. Further, rabbitsimmunized with constructs containing deletions of the V2 regiongenerally generated similar antibody titers relative to rabbitsimmunized with the companion construct still containing the V2 region.

The nucleic acid immunizations are followed by protein boosting witho-gp140.modSF162.delV2 (0.1 mg of purified protein) at 24 weeks afterthe initial immunization. Results are shown in Table 16.

TABLE 16 GMT 2 wks GMT 2 wks GMT 2 wks post- post-2nd DNA post-3rd DNAprotein Group immunization immunization boost gp120.modSF162 4573 589926033 gp120.modSF162.delV2 3811 3122 29606 gp140.modSF162 1478 710 12882gp140.modSF162.delV2 1572 819 11067 gp140.mut.modSF162 1417 788 8827gp140.mut.modSF162.delV2 1378 1207 13301 gp160.modSF162 23 81 7050gp160.modSF162.delV2 85 459 11568

All constructs are highly immunogenic and generate substantial antigenbinding antibody responses after only 2 immunizations in rabbits.

C. Baboons

Groups of four baboons were immunized intramuscularly with 1 mg doses ofDNA encoding different forms of synthetic US4 gp140 (see the followingtable) at 0, 4, 8, 12, 28, and 44 weeks. The animals were also boostedtwice with US4 o-gp140 protein (gp140.mut.modUS4) at 44 and 76 weeksusing MF59 as adjuvant. Results are shown in Table 17.

TABLE 17 2 Wks post 2 Wks post 7th DNA (o- 2 Wks Post 6th DNA gp140 5thDNA (plus o- protein Animal Treatment immunization gp140 prot.immuniz.)only) CY 215 gp140.modUS4 8.3 446 1813 CY 216 8.3 433 1236 CY 217 681660 2989 CY 218 101 2556 1610 Geomean: 26.2 951.4 1812.1 CY 219gp140.modUS4 + 8.3 8.3 421 CY 220 p55gag.SF2 8.3 8.3 3117 CY 221 8.3 954871 CY 222 8.3 71 916 Geomean: 8.3 46.5 1011.5 CY 223 gp140.mut.modUS441.4 10497 46432 CY 224 8.3 979 470 CY 225 135 2935 3870 CY 226 47 12094009 Geomean: 68.3 2457.4 4289.6 CY 227 gp140TM.modUS4 8.3 56 5001 CY228 8.3 806 1170 CY 229 8.3 48 3402 CY 230 8.3 38 6520 GMT*: 8.3 95.33375.3 *GMT = geometric mean titer

The results in Table 17 demonstrate the usefulness of the syntheticconstructs to generate immune responses in primates such as baboons. Inaddition, all animals showed evidence of antigen-specific (Env antigen)lymphopro-liferative responses.

D. Rhesus Macaques

Two rhesus macaques (designated H445 and J408) were immunized with 1 mgof DNA encoding SF162 gp140 with a deleted V2 region (SF162.9p140.delV2)by intramuscular (IM) and intradermal (ID) routes at 0, 4, 8, and 28weeks. Approximately 100 μg of the protein encoded by the SF162.gp140mut.delV2 construct was also administered in MF59 by IM delivery at28 weeks.

ELISA titers are shown in FIG. 61. Neutralizing antibody activity isshown Tables 18 and 19. Neutralizing antibody activity was determinedagainst a variety of primary HIV-1 isolates in a primary lymphocyte or“PBMC-based” assay (see the following tables). Further, the phenotypicco-receptor usage for each of the primary isolates is indicated. As canbe seen in the tables neutralizing antibodies were detected againstevery isolate tested, including the HIV-1 primary isolates (i.e.,SF128A, 92US660, 92HT593, 92US657, 92US714, 91US056, and 91US054).

TABLE 18 Treatment Bleed 0 Bleed 1 Bleed 2 1st 2nd 1st 2nd 2 Wks AnimalImmunization Immunization Imm'n Imm'n post 2nd EO 456 25 μg 120mod(None) 8.3 45 309 EO 457 DNA 8.3 254 460 EO 458 8.3 8.3 93 EO 459 8.3 4345 EO 460 8.3 8.3 274 EO 461 25 μg 120mod 25 μg 120mod 8.3 47 1502 EO462 DNA DNA 8.3 80 5776 EO 463 8.3 89 3440 EO 464 8.3 8.3 3347 EO 4658.3 69 1127 EO 466 50 μg 120mod (None) 8.3 63 102 EO 467 DNA 8.3 112 662EO 468 8.3 94 459 EO 469 8.3 58 48 EO 470 8.3 95 355 EO 471 50 μg 120mod50 μg 120mod 8.3 110 9074 EO 472 DNA DNA 8.3 8.3 4897 EO 473 8.3 49 4089EO 474 8.3 59 5280 EO 475 8.3 8.3 929 EO 476 25 μg 120mod Sindbis/Env8.3 653 EO 477 DNA 8.3 87 22675 EO 478 8.3 76 3869 EO 479 8.3 1004 EO480 8.3 71 7080

TABLE 19 Treatment Bleed 0 Bleed 1 Bleed 2 1st 2nd 1st 2nd 2 Wks AnimalImmunization Immunization Imm'n Imm'n post 2nd EO 481 Sindbis/Env (None)8.3 8.3 8.3 EO 482 8.3 8.3 8.3 EO 483 8.3 78 103 EO 484 8.3 8.3 32 EO485 8.3 76 207 EO 486 Sindbis/Env Sindbis/Env 8.3 8.3 458 EO 487 8.3 8.3345 EO 488 8.3 8.3 331 EO 489 8.3 103 111 EO 490 8.3 8.3 5636

Lymphoproliferative activity (LPA) was also determined by antigenicstimulation followed by uptake of ³H-thymidine in these animals and isshown in Table 20. Experiment 1 was performed at 14 weeks post third DNAimmunization and Experiment 2 was performed at 2 weeks post fourth DNAimmunization using DNA and protein. For gp120ThaiE, gp120SF2 and US4o-gp140, appropriate background values were used to calculateStimulation Indices (S. I.; Antigenic stimulation CPM/Background CPM).

TABLE 20 S.I.: Calculated as Ag CPM/Background CPM Animal/exp#gp120ThaiE gp120 SF2 env2-3SF2 o-gp140US4 J408/#1 2 1 1 5 H445/#1 1 1 16 J408/#2 1 1 2 3 H445/#2 0 0 3 2

As can be seen by the results presented in Table 20 lymphoproliferativeresponses to o-gp140.US4 antigen were also in all four animals at bothexperimental time points. Such proliferation results are indicative ofinduction of T-helper cell functions.

The results presented above demonstrate that the syntheticgp140.modSF162.delV2 DNA and protein are immunogenic in non-humanprimates.

Example 13 In Vitro Expression of Recombinant Sindbis RNA and DNAContaining the Synthetic Gag or Env Expression Cassettes A. SyntheticGag Expression Cassettes

To evaluate the expression efficiency of the synthetic Gag expressioncassette in Alphavirus vectors, the synthetic Gag expression cassettewas subcloned into both plasmid DNA-based and recombinant vectorparticle-based Sindbis virus vectors. Specifically, a cDNA vectorconstruct for in vitro transcription of Sindbis virus RNA vectorreplicons (pRSIN-luc; Dubensky, et al., J Virol. 70:508-519, 1996) wasmodified to contain a PmeI site for plasmid linearization and apolylinker for insertion of heterologous genes. A polylinker wasgenerated using two oligonucleotides that contain the sites XhoI, PmlI,ApaI, NarI, XbaI, and NotI (XPANXNF, SEQ ID NO:17, and XPANXNR, SEQ IDNO:18).

The plasmid pRSIN-luc (Dubensky et al., supra) was digested with XhoIand NotI to remove the luciferase gene insert, blunt-ended using Klenowand dNTPs, and purified from an agarose get using GeneCleanII (Biol0l,Vista, Calif.). The oligonucleotides were annealed to each other andligated into the plasmid. The resulting construct was digested with NotIand SacI to remove the minimal Sindbis 3′-end sequence and A₄₀ tract,and ligated with an approximately 0.4 kbp fragment from PKSSIN-BV (WO97/38087). This 0.4 kbp fragment was obtained by digestion of pKSSIN-BVwith NotI and SacI, and purification after size fractionation from anagarose gel. The fragment contained the complete Sindbis virus 3′-end,an A₄₀ tract and a PmeI site for linearization. This new vectorconstruct was designated SINBVE.

The synthetic HIV Gag coding sequence was obtained from the parentalplasmid by digestion with EcoRI, blunt-ending with Klenow and dNTPs,purification with GeneCleanII, digestion with Sail, size fractionationon an agarose gel, and purification from the agarose gel usingGeneCleanII. The synthetic Gag coding fragment was ligated into theSINBVE vector that had been digested with XhoI and PmlI. The resultingvector was purified using GeneCleanII and designated SINBVGag. VectorRNA replicons may be transcribed in vitro (Dubensky et al., supra) fromSINBVGag and used directly for transfection of cells. Alternatively, thereplicons may be packaged into recombinant vector particles byco-transfection with defective helper RNAs or using an alphaviruspackaging cell line as described, for example, in U.S. Pat. Nos.5,843,723 and 5,789,245, and then administered in vivo as described.

The DNA-based Sindbis virus vector pDCMVSIN-beta-gal (Dubensky, et al.,J. Virol. 70:508-519, 1996) was digested with Sail and XbaI, to removethe beta-galactosidase gene insert, and purified using GeneCleanII afteragarose gel size fractionation. The HIV Gag gene was inserted into thepDCMVSIN-beta-gal by digestion of SINBVGag with SalI and XhoI,purification using GeneCleanII of the Gag-containing fragment afteragarose gel size fractionation, and ligation. The resulting constructwas designated pDSIN-Gag, and may be used directly for in vivoadministration or formulated using any of the methods described herein.

BHK and 293 cells were transfected with recombinant Sindbis vector RNAand DNA, respectively. The supernatants and cell lysates were testedwith the Coulter p24 capture ELISA (Example 2).

BHK cells were transfected by electroporation with recombinant SindbisRNA. The expression of p24 (in ng/ml) is presented in Table 21. In thetable, SINGag#1 and 2 represent duplicate measurements, and SINβgalrepresents a negative control. Supernatants and lysates were collected24 h post transfection.

TABLE 21 Construct Supernatant Lysate SINβgal RNA 0  0 SINGag#1 RNA 7 ngMax (approx. 1 μg) SINGag#2 RNA 1 ng 700 ng

293 cells were transfected using LT-1 (Example 2) with recombinantSindbis DNA. Synthetic pCMVKM2GagMod.SF2 was used as a positive control.Supernatants and lysates were collected 48 h post transfection. Theexpression of p24 (in ng/ml) is presented in Table 22.

TABLE 22 Construct Supernatant Lysate SINGag DNA 3 30 pCMVKM2.GagMod.SF232 42 DNA

The results presented in Tables 21 and 22 demonstrate that Gag proteinscan be efficiently expressed from both DNA and RNA-based Sindbis vectorsystems using the synthetic Gag expression cassette (p55Gag.mod).

B. Synthetic Env Expression Cassettes

To evaluate the expression efficiency of the synthetic Env expressioncassette in Alphavirus vectors, synthetic Env expression cassettes weresubcloned into both plasmid DNA-based and recombinant vectorparticle-based Sindbis virus vectors as described above for Gag.

The synthetic HIV Env coding sequence was obtained from the parentalplasmid by digestion with SalI and XbaI, size fractionation on anagarose gel, and purification from the agarose gel using GeneCleanII.The synthetic Env coding fragment was ligated into the SINBVE vectorthat had been digested with XhoI and XbaI. The resulting vector waspurified using GeneCleanII and designated SINBVEnv. Vector RNA repliconsmay be transcribed in vitro (Dubensky et al., supra) from SINBVEnv andused directly for transfection of cells. Alternatively, the repliconsmay be packaged into recombinant vector particles by co-transfectionwith defective helper RNAs or using an alphavirus packaging cell lineand administered as described above for Gag.

The DNA-based Sindbis virus vector pDCMVSIN-beta-gal (Dubensky, et al.,J. Virol. 70:508-519, 1996) was digested with Sail and XbaI, to removethe beta-galactosidase gene insert, and purified using GeneCleanII afteragarose gel size fractionation. The HIV Env gene was inserted into thepDCMVSIN-beta-gal by digestion of SINBVEnv with XbaI and XhoI,purification using GeneCleanII of the Env-containing fragment afteragarose gel size fractionation, and ligation. The resulting constructwas designated pDSIN-Env, and may be used directly for in vivoadministration or formulated using any of the methods described herein.

BHK and 293 cells were transfected with recombinant Sindbis vector RNAand DNA, respectively. The supernatants and cell lysates were tested bycapture ELISA.

BHK cells were transfected by electroporation with recombinant SindbisRNA. The expression of Env (in ng/ml) is presented in Table 23. In thetable, the Sindbis RNA containing synthetic Env expression cassettes areindicated and βgal represents a negative control. Supernatants andlysates were collected 24 h post transfection.

TABLE 23 Supernatant Lysate Construct (Neat) ng/ml (1:10 dilution) ng/mlβgal RNA 0 0 gp140.modUS4 726 7147 gp140.modSF162 3529 7772gp140.modUS4.delV1/V2 1738 6526 gp140.modUS4.delV2 960 3023gp140.modSF162.delV2 2772 3359

293 cells were transfected using LT-1 mediated transfection (PanVera)with recombinant Sindbis DNA containing synthetic expression cassettesof the present invention and βgal sequences as a negative control.Supernatants and lysates were collected 48 h post transfection. Theexpression of Env (in ng/ml) is presented in Table 24.

TABLE 24 Lysate Supernatant (1:10 Construct (Neat) ng/ml dilution) ng/mlβgal 0 0 gp140.modSF162.delV2 1977 801 gp140.modSF162 949 746

The results presented in Tables 23 and 24 demonstrated that Env proteinscan be efficiently expressed from both DNA and RNA-based Sindbis vectorsystems using the synthetic Env expression cassettes of the presentinvention.

Example 14 A. In vivo Immunization with Gag-containing DNA and/orSindbis Particles

CB6F1 mice were immunized intramuscularly at 0 and 4 weeks with plasmidDNA and/or Sindbis vector RNA-containing particles each containingGagMod.SF2 sequences as indicated in Table 25. Animals were challengedwith recombinant vaccinia expressing SF2 Gag at 3 weeks post secondimmunization (at week 7). Spleens were removed from the immunized andchallenged animals 5 days later for a standard ⁵¹C release assay for CTLactivity. Values shown in Table 25 indicate the results from the spleensof three mice from each group. The boxed values in Table 25 indicatethat all groups of mice receiving immunizations with pCMVKm2.GagMod.SF2DNA and/or SindbisGagMod.SF2 virus particles either alone or incombinations showed antigen-specific CTL activity.

TABLE 25 Cytotoxic T-lymphocyte (CTL) responses in mice immunized withHIV-1 gagmod DNA and Sindbis gagmod virus particles

a 20 μg b 10⁷ particles release assay. * Challenge with recombinantvaccinia virus expressing HIV-1SF2 Gag at 3 weeks post secondimmunization (week 7). Spleens taken 5 days later. Ex vivo CTL assayperformed by standard ⁵¹Cr release assay. Values seen represent resultsfrom 3 pooled mouse spleens per group

B. In Vivo Immunization with Env-Containing DNA and/or Sindbis Particles

Balb/C mice were immunized intramuscularly at 0 and 4 weeks(as shown inthe following table) with plasmid DNA and/or Sindbis-virusRNA-containing particles each containing gp120.modUS4 sequences.Treatment regimes and antibody titers are shown in Table 26. Antibodytiters were determined by ELISA using gp120 SF2 protein to coat theplates.

TABLE 26 Bleed 1 Bleed 2 Treatment Bleed 0 (8 wks) (10 wks) 1st 2nd 1st2nd 2 Wks Animal Immunization Immunization Imm'n Imm'n post 2nd EO 45625 μg 120mod (None) 8.3 45 309 EO 457 DNA 8.3 254 460 EO 458 8.3 8.3 93EO 459 8.3 43 45 EO 460 8.3 8.3 274 EO 461 25 μg 120mod 25 μg 120mod 8.347 1502 EO 462 DNA DNA 8.3 80 5776 EO 463 8.3 89 3440 EO 464 8.3 8.33347 EO 465 8.3 69 1127 EO 466 50 μg 120mod (None) 8.3 63 102 EO 467 DNA8.3 112 662 EO 468 8.3 94 459 EO 469 8.3 58 48 EO 470 8.3 95 355 EO 47150 μg 120mod 50 μg 120mod 8.3 110 9074 EO 472 DNA DNA 8.3 8.3 4897 EO473 8.3 49 4089 EO 474 8.3 59 5280 EO 475 8.3 8.3 929 EO 476 25 μg120mod Sindbis/Env 8.3 653 EO 477 DNA 8.3 87 22675 EO 478 8.3 76 3869 EO479 8.3 1004 EO 480 8.3 71 7080 EO 481 Sindbis/Env (None) 8.3 8.3 8.3 EO482 8.3 8.3 8.3 EO 483 8.3 78 103 EO 484 8.3 8.3 32 EO 485 8.3 76 207 EO486 Sindbis/Env Sindbis/Env 8.3 8.3 458 EO 487 8.3 8.3 345 EO 488 8.38.3 331 EO 489 8.3 103 111 EO 490 8.3 8.3 5636

As can be seen from the data presented above, all of the mice generallydemonstrated substantial immunological responses by bleed number 2. ForEnv, the best results were obtained using either (i) 50 μg ofgp120.modUS4 DNA for the first immunization followed by a secondimmunization using 50 μg of gp120.modUS4 DNA, or (ii) 25 μg ofgp120.modUS4 DNA for the first immunization followed by a secondimmunization using 10⁷ pfus of Sindbis.

The results presented above demonstrate that the Env and Gag proteins ofthe present invention are effective to induce an immune response usingSindbis vector systems which include the synthetic Env (e.g.,gp120.modUS4) or Gag expression cassettes.

Example 15 Co-Transfection of Env and Gag as Monocistronic andBicistronic Constructs

DNA constructs encoding (i) wild-type US4 and SF162 Env polypeptides,(ii) synthetic US4 and SF162 Env polypeptides (gp160.modUS4,gp160.modUS4.delV1/V2, gp160.modSF162, and gp120.modSF162.delV2), and(iii) SF2-gag polypeptide (i.e., the Gag coding sequences obtained fromthe SF2 variant or optimized sequences corresponding to thegagSF2-gag.modSF2) were prepared. These monocistronic constructs wereco-transfected into 293T cells in a transient transfection protocolusing the following combinations: gp160.modUS4; gp160.modUS4 andgag.modSF2; gp160.modUS4.delV1/V2; gp160.modUS4.delV1/V2 and gag.modSF2;gp160.modSF162 and gag.modSF2; gp120.modSF162.delV2 and gag.modSF2; andgag.modSF2 alone.

Further several bicistronic constructs were made where the codingsequences for Env and Gag were under the control of a single CMVpromoter and, between the two coding sequences, an IRES (internalribosome entry site (EMCV TRES); Kozak, M., Critical Reviews dnBiochemistry and Molecular Biology 27(45):385-402, 1992; Witherell, G.W., et al., Virology 214:660-663, 1995) sequence was introduced afterthe Env coding sequence and before the Gag coding sequence. Thoseconstructs were as follows: gp160.modUS4.gag.modSF2, SEQ ID NO:73 (FIG.61); gp160.modUSF162.gag.modSF2, SEQ ID NO:74 (FIG. 62);gp160.modUS4.delV1/V2.gag.modSF2, SEQ ID NO:75 (FIG. 63); andgp160.modSF162.delV2.gag.modSF2, SEQ ID NO:76 (FIG. 64).

Supernatants from cell culture were filtered through 0.45 μm filtersthen ultracentrifuged for 2 hours at 24,000 rpm (140,000×g) in an SW28rotor through a 20% sucrose cushion. The pelleted materials weresuspended and layered on a 20-60% sucrose gradient and spun for 2 hoursat 40,000 rpm (285,000×g) in an SW41Ti rotor. Gradients werefractionated into 1.0 ml samples. A total of 9-10 fractions weretypically collected from each DNA transfection group.

The fractions were tested for the presence of the Env and Gag proteins(across all fractions). These results demonstrated that the appropriateproteins were expressed in the transfected cells (i.e., if an Env codingsequence was present the corresponding Env protein was detected; if aGag coding sequence was present the corresponding Gag protein wasdetected).

Virus like particles (VLPs) were known to be present through a selectedrange of sucrose densities. Chimeric virus like particles (VLPs) wereformed using all the tested combinations of constructs containing bothEnv and Gag. Significantly more protein was found in the supernatantcollected from the cells transfected with “gp160.modUS4.delV1/V2 andgag.modSF2” than in all the other supernatants.

Western blot analysis was also performed on sucrose gradient fractionsfrom each transfection. The results show that bicistronic plasmids gavelower amounts of VLPs than the amounts obtained using co-transfectionwith monocistronic plasmids.

In order to verify the production of chimeric VLPs by these cell linesthe following electron microscopic analysis was carried out.

293T cells were plated at a density of 60-70% confluence in 100 mmdishes on the day before transfection. The cells were transfected with10 μg of DNA in transfection reagent LT1 (Panvera Corporation, 545Science Dr., Madison, Wis.). The cells were incubated overnight inreduced serum medium (opti-MEM, Gibco-BRL, Gaithersburg, Md.). Themedium was replaced with 10% fetal calf serum, 2% glutamine in IMDM inthe morning of the next day and the cells were incubated for 65 hours.Supernatants and lysates were collected for analysis as described above(see Example 2).

The fixed, transfected 293T cells and purified ENV-GAG VLPs wereanalyzed by electron microscopy. The cells were fixed as follows. Cellmonolayers were washed twice with PBS and fixed with 2% glutaraldehyde.For purified VLPs, gradient peak fractions were collected andconcentrated by ultracentrifugation (24,000 rpm) for 2 hours. Electronmicroscopic analysis was performed by Prof. T. S. Benedict Yen (VeteransAffairs, Medical Center, San Francisco, Calif.).

Electron microscopy was carried out using a transmission electronmicroscope (Zeiss 10c). The cells were pre-stained with osmium andstained with uranium acetate and lead citrate. Immunostaining wasperformed to visualize envelope on the VLP. The magnification was100,000×.

FIGS. 65A-65F show micrographs of 293T cells transfected with thefollowing constructs: FIG. 65A, gag.modSF2; FIG. 65B, gp160.modUS4; FIG.65C, gp160.modUS4.delV1/V2.gag.modSF2 (bicistronic Env and Gag); FIGS.65D and 65E, gp160.modUS4.delV1/V2 and gag.modSF2; and FIG. 65F,gp120.modSF162.delV2 and gag.modSF2. In the figures, free and buddingimmature virus-like-particles (VLPs) of the expected size (approximately100 nm) decorated with the Env protein were seen. In sum, gp160polypeptides incorporate into Gag VLPs when constructs wereco-transfected into cells. The efficiency of incorporation is 2-3 foldhigher when constructs encoding V-deleted Env polypeptides from highsynthetic expression cassettes are used.

Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. A method for producing virus-like particles (VLPs), comprisingincubating a cell under conditions for producing the VLPs, wherein thecell comprises an expression cassette, wherein the expression cassettecomprises a polynucleotide sequence encoding a polypeptide including anHIV Gag polypeptide, wherein the polynucleotide sequence encoding theGag polypeptide comprises a sequence having at least 90% sequenceidentity to a nucleotide sequence selected from the group consisting ofSEQ ID NO: 9 and SEQ ID NO:4, and wherein the polynucleotide sequence isoperably linked to control elements compatible with expression in thecells.
 2. The method of claim 1 wherein the polynucleotide sequencefurther includes a polynucleotide sequence encoding an HIV polypeptideselected from the group consisting of: a protease polypeptide; a reversetranscriptase polypeptide; a tat polypeptide; a core polypeptide,wherein the polynucleotide sequence encoding the core polypeptide (1)has at least 90% identity to the nucleotide sequence SEQ ID NO:4 and (2)is modified by deletions of coding regions corresponding to reversetranscriptase and integrase; a core polypeptide, wherein thepolynucleotide sequence encoding the core polypeptide has at least 90%identity to the nucleotide sequence SEQ ID NO:7; and a polymerasepolypeptide, wherein the polynucleotide sequence encoding the polymerasepolypeptide comprises a nucleotide sequence having at least 90% identityto the nucleotide sequence SEQ ID NO:6.
 3. The method of claim 2 whereinthe polynucleotide sequence encodes the protease polypeptide andcomprises a nucleotide sequence having at least 90% identity to anucleotide sequence selected from the group consisting of SEQ ID NO:5,SEQ ID NO:78, and SEQ ID NO:79.
 4. The method of claim 2 wherein thepolynucleotide sequence encodes the reverse transcriptase polypeptideand comprises a nucleotide sequence having at least 90% identity to anucleotide sequence selected from the group consisting of SEQ ID NO:80,SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.
 5. Themethod of claim 2 wherein the polynucleotide sequence encodes the tatpolypeptide and comprises a nucleotide sequence having at least 90%identity to a nucleotide sequence selected from the group consisting ofSEQ ID NO:88 and SEQ ID NO:89.
 6. The method of claim 1 wherein the cellis selected from: the group consisting of BHK, VERO, HT1080, 293, RD,COS-7, CHO cells, and insect cells; the group consisting of a bacterialcell, a yeast cell, a plant cell, and an antigen presenting cell; andthe group consisting of an immortalized cell and a tumor-derived cell.8. The method of claim 1 further comprising substantially purifying theVLPs to produce a composition of VLPs.